Broadly Neutralizing Anti-HIV Antibodies and Epitope Therefor

ABSTRACT

The present invention relates to broadly neutralizing anti-HIV-1 antibodies and isolated antigens. Also disclosed are related methods and compositions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.61/1934,359 filed Jan. 31, 2014, the contents of which are incorporatedherein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

The invention disclosed herein was made, at least in part, withGovernment support under. Grant Nos. HIVRAD P01 A1100148 and 1UM1AI100663-01 from the National Institutes of Health. Accordingly, theU.S. Government has certain s in this invention.

BACKGROUND OF THE INVENTION

The only target of neutralizing anti-HIV-1 antibodies is the envelope(Env) spike, a heterotrimer of gp120 and gp41 subunits. Singlecell-based antibody cloning techniques have recently uncovered a largenumber of antibodies that can potently neutralize highly diverse HIV-1variants by targeting Env (Klein et al., Science 341, 1199 (2013)). Whentransferred passively, broadly neutralizing antibodies (bNAbs) canprevent infection by HIV-1 or SHIV in humanized mice and macaques,respectively. Moreover, combinations of bNAbs can also suppressestablished V-1 and SHIV infections (Klein et al., Nature 492, 118(2012); Barouch et al., Nature 503, 224 (2013); Shingai et al., Nature503, 277 (2013)).

Most of the bNAbs characterized to date target one of four major sitesof vulnerability on HIV-1 Env: on gp120, the CD4 binding site, the V2loop, and the base of the V3 loop, and on gp41, the membrane proximalregion (Klein et al., Science 341, 1199 (2013), Burton et al., Science337, 183 (2012); Mascola et al., Immunological Reviews 254, 225 (2013)).8ANC195 is among a small group of bNAbs that does not appear to targetany of these sites. Although only two of the B cells originally isolatedfrom the 8ANC195 donor, an. HIV-1 elite controller, belonged to the8ANC195 clone, the antibodies produced by this clone complemented theneutralizing activity of antibodies produced by a more expanded. B cellclone that targeted the CD4 binding site (Scheid et al., Science 333,1633 (2011)).

8ANC1.95 is classified as a bNAb because it neutralized 66% of virusesin a diverse viral panel. (Scheid. et al.. Science 333 1633 (201 1)).Like other anti-HIV-1 bNAbs, 8ANC195 is highly somatically mutated,including insertions and deletions in the complementarily determiningregions (CDRs) and framework regions (FWRs) of its heavy chain (HC) andlight chain (LC). Although initial efforts to map the 8ANC195 epitopewere unsuccessful (Ibid.) computational analyses of neutralization datapredicted that intact potential N-linked glycosylation sites (PNGSs) atpositions 234_(gp120) and 276_(gp120) were essential for its activity.These predictions were confirmed by evaluating the neutralizationpotency of 8ANC195 against mutant HIV-1 strains in vitro and in vivo(West, jr. et al., Proceedings of the National Academy of Sciences ofthe United States America 110, 10598 (2013): Chuang et al. Journal ofVirology 87, 10047 (2013)). However, the precise 8ANC195 epitope onHIV-1 Env has heretofore remained elusive.

SUMMARY OF INVENTION

This invention relates, in part, to the isolation f broadly-neutralizingantibodies (bNAbs) directed at an epitope on the HIV-1 envelope spikethat spans the gp120 and gp41 subunits.

In one embodiment, the antibody comprises a heavy chain having one ofthe following amino acid sequences:

(g52; SEQ ID NO: 1) QIHLVQSGTEVKKPGSSVTVSCKAYGVNTFGLYAVNWVRQAPGQSLEYIGQIWRWKSSASHHFRGRVLISAVDLTGSSPPISSLEIKNLTSDDTAVYFCTTTSTYDRWSGLHHDGVMAFSSWGQGTLISVSAASTKG; (g23; SEQ ID NO: 2)QIHLVQSGTEVKKPGSSVTVSCKAYGVNTFGLYAVNWVRQAPGQSLEYIGQIWRWKSSASHHFRGRVIISAVDLTGSSPPISSLEIKNLTSDDTAVYFCTTTSTSDYWSGLHHDGVMAFSSWGQGTLISVSAASTKG; (g8; SEQ ID NO: 3)QIHLVQSGTGVKKPGSSVTVSCKAYGVNTFGLYAVNWVRQAPGQGLEYIGQIWRWKSSASHHFRGRVLISAVDLTGSSPPITSLEIKNVTSDDTAVYFCTTTSTYDKWSGLYHDGVMAFSSWGQGTLISVSAASTKG; (g20; SEQ ID NO: 4)QIHLVQSGTEVKKPGSSVAVSCKAYGVNTFGLYAVNWVRQAPGQSLEYIGQIWRWKSSASHDFRGRVIISAVDLTGSSPPISSLEIKNLTSDDTAVYFCTATSTPDYWSGLHHDGVMAFSSWGQGTLISVSAASTKG; (g59; SEQ ID NO: 5)QIHLVQSGTEVKKPGSSVTVSCKAYGVNTFGLYAVNWVRQAPGQGLEYIGQIWRWKSSASHHFRGRVLISAVDLTGSSPPISSLEIKNVTSDDTAVYFCTTTSTYDEWSDLHHDGVMAFSSWGQGTLISVSAASTKG; (g62; SEQ ID NO: 6)QIHLVQSGTEVKKPGSSVTVSCKAYGVNTFGLYAVNWVRQAPGQSLEYIGQIWRWKSSASHHFRGRVLISAVDLTGSSPPISSLEIKNLTSDDTAVYFCTTTSTYDKWSGLHHDGVMAFSSRGQGTLISVSAASTKG; (g22; SEQ ID NO: 7)QIHLVQSGTEVKKPGSSVTVSCKAYGVNTFGLYAVNWVRQAPGQSLEYIGQIWRWKSSASHHFRGRVLISAVDLTGPSPPISSLEIKNLTSDDTAVYFCTTTSTYDKWSGLHHDGVMAFSSWGQGTLISVSAASTKG; (g15; SEQ ID NO: 8)QIHLVQSGTEVKKPGSSVTVSCKAYGVNTFGLYAVNWVRQAPGQSLEYIGQIWRWKSSASHHFRGRVIISAVDLTGSSPPISSLEIKNLTSDDTAVYFCTTASTYDKWSGLHHDGVMAFSSWGQGTLISVSAASTKG; (g4; SEQ ID NO: 9)QIHLVQSGTEVKKPGSSVTVSCKAYGVNTFGLYAVNWVRQAPGQSLEYIGQIWRWKSSASHHFRGRVIISAVDLTGSSPPISPLEIKNLTSDDTAVYFCTTTSTSDRWSGLHHDGVMAFSSWGQGTLISVSAASTKG; (g46; SEQ ID NO: 10)QIHLVQSGTEVKKPGSSVTVSCKAYGVNTFGLYAVNWVRQAPGQGLEYIGQIWRWKSSASHHFRGRVLISAVDLTGSSPPISSLEIKNVTSDDTAVYFCTTTSTYDKWSGLHHDGVVAFSSWGQGTLISVSAASTKG; (g44; SEQ ID NO: 11)QIHLVQSGTEVKKPGSSVTVSCKAYEVNTFGLYAVNWVRQAPGQSLEYIGQIWRWKSSASHHFRGRVLISAVDLTGSSPPISSLEIKNVTSDDTAVYFCTTTSTHDKWSGLHHDGVMAFSSWGQGTLISVSAASTKG; (g50; SEQ ID NO: 12)QIHLVQSGTEVKKPGSSVTVSCKAYGVNTFGLYAVNWVRQAPGQGLEYIGQIWRWKSSASHHFRGRVLISAIDLTGSSPPISSLEIKNVTSDDTAVYFCTTMSTYDKWSGLHHDGVMAFSSWGQGTLISVSAASTKG; (g3; SEQ ID NO: 13)QIHLVQSGTEVKKPGSSVTVSCKAYGVNTFGLYAVSWVRQAPGQRLEYIGQIRRWKSSASHHFRGRVTVSAVDPTGSSPPISSLEIRDLTTDDTAVYFCTTTSTSDYWSGLHNERGTAFSSWGQGTLISVSAASTKG; (3040HC; SEQ ID NO: 14)QIHLVQSGTEVKKPGSSVTVSCKAYGVNTFGLYAVNWVRQAPGQSLEYIGQIWRWKSSASHHFRGRVLISAVDLTGSSPP1SSLEIKNLTSDDTAVYFCTTTSTYDQWSGLHHDGVMAFSSWGQGTLISVSAASTKG; (3430HC; SEQ ID NO: 15)QIHLVQSGTEVKKPGSSVTVSCKAYGVNTFGLYAVNWVRQAPGQSLEYIGQIWRWKSSASHHFRGRVIISAVDLTGSSPPISSLEIKNLTSDDTAVYFCTTTSTSDYWSGLHHDGVMAFSSWGQGTLISVSAASTKG; (3484HC; SEQ ID NO: 16)QIHLVQSGTEVKKPGSSVTVSCKAYGVNTFGLYAVNWVRQAPGQSLEYIGQIWRWKSSASHHFRGRVLISAVDLTGSSPPISSLEIKNLTSDDTAVYFCTTTSTYDRWSGLHHDGVMAFSSWGQGTLISVSAASTKG; (3044HC: SEQ ID NO: 17)QIHLVQSGTEVRKPGSSVTVSCKAYGVNTFGLYAVNWVRQAPGQSLEYIGQIWRWKSSASHHFRGRVLISAVDLTGSSPPISSLEIKNLTSDDTAVYFCTTTSTYDKWSGLHHDGVMAFSSWGQGTLISVSAASTKG; and (3630HC: SEQ ID NO: 18)QIHLVQSGTEVKKPGSSVTVSCKAYGVNTFGLYAVNWVRQAPGQSLEYIGQIWRWKSSASHHFRGRVLISAVDLTGSSPPISSLEIKNLTSDDTAVYFCTTTSTYDRWSGLHHDGVMAFSSWGQGTLISVSAASTKG.

In one embodiment, the antibody comprises a light chain having one ofthe following amino acid sequences:

(k3; SEQ ID NO: 19) DIQMTQSPSTLSASIGDTVRISCRASQSITGNWLAWYHQRPGKAPRLLIYRGSRLLGGVPSRFSGSAAGTDFTLTIANLQAEDFGTFYCQQYDTYPGTFG QGTKVEVKRTVAAPSVF;(k5; SEQ ID NO: 20) DIQMTQSPSTLSASTGDTVRISCRASQSITGNWVAWYQQRPGKAPRLLIYRGAALLGGVPSRFRGSAAGTDFTLTIGNLQAEDFGTFYCQQYDTYPGTFG QGTKVEVKRTVAAPSVF;(k59; SEQ ID NO: 21) DIQMTQSPSTLSASIGDTVRISCRASQSITGGWLAWYHQRPGKAPRLLIYRGSRLLGGVPSKFSGSAAGTDFTLTIANLQAEDFGTFYCQQYDTYPGTFG QGTKVEVKRTVAAPSVF;(k62; SEQ ID NO: 22) DIQMTQSPSTLSASIGDTVRISCRASQSITGGWLAWYHQRPGKAPRLLIYRGSRLVGGVPSRFSGSAAGTDFTLTIGNLQAEDFGTFYCQQYDTYPGTFG QGTKVEVKRTVAAPSVF;(k18; SEQ ID NO: 23) DIQMTQSPSTLSASVGDTVRISCRASQSITGGWLAWYHQRPGKAPRLLIYRGSRLLGGVPSRFSGSAAGADFTLTIANLQAEDFGTFYCQQYDTYPGTFG QGTKVEVKRTVAAPSVF;(k53; SEQ ID NO: 24) DIQMTQSPSTLSASIGDTVMISCRASQSITGGWLAWYHQRPGKAPRLLIYRGSKLLGGVPSRFSGSAAGTGFTLTIGNLQAEDFGTFYCQQYDTYPGTFG QGTKVEVKRTVAAPSVF;(k61; SEQ ID NO: 25) DIQMTQSPSTLSASIGDTVRISCRASQSITGNWVAWYHQRPGKAPRLLIYRGAALLGGVPSRFSGSAAGTDFTLTIGNLQAEDFGTFYCQQYDTYPGTFG QGTKVEVKRTVAAPSVF;(k11; SEQ ID NO: 26) DIQMTQSPSTLSASVGGTVRISCRASQSITGGWLAWYHQRPGKAPRLLIYRGSRLLGGVPSRFSGSAAGTDFTLTIANLQAEDFGTFYCQQYDTYPGTFG QGTKVEVKRTVAAPSVF;(k19; SEQ ID NO: 27) DIQMTQSPSTLSASVGDTVRISCRASQSITGGWLAWYHQRPGKAPRLLIYRGSRLLGGVPSRFSGSAAGTGFTLTIANLQAEDFGTFYCQQYDTYPGTFG QGTKVEVKRTVAAPSVF;(k81; SEQ ID NO: 28) DIQMTQSPSTLSASIGDTVRISCRASQSITGGWVAWYHQRPGKAPRLLIYRGSRLLGGVPSRFSGSAAGTDFTLTIGNLQAEDFGTFYCQQYDTYPGTFG QGTKVEVKRTVAAPSVF (3040LC; SEQ ID NO: 29)DIQMTQSPSTLSASIGDTVRISCRASQSITGNWVAWYQQRPGKAPRLLIYRGAALLGGVPSRFSGSAAGTDFTLTIGNLQAEDFGTFYCQQYDTYPGTFG QGTKVEVKRTVAAPSVF;(3430LC; SEQ ID NO: 30)DIQMTQSPSTLSASVGDTVRISCRASQSITGGWLAWYHQRPGKAPRLLIYRGSRLLGGVPSRFSGSAAGTDFTLTIANLQAEDFGTFYCQQYDTYPGTFG QGTKVEVKRTVAAPSVF;(3484LC; SEQ ID NO: 31)DIQMTQSPSTLSASIGDTVRISCRASQSITGNWVAWYQQRPGKAPRLLIYRGAALLGGVPSRFRGSAAGTDFTLTIGNLQAEDFGTFYCQQYDTYPGTFG QGTKVEVKRTVAAPSVF;(3044LC; SEQ ID NO: 32)DIQMTQSPSTLSASIGDTVRISCRASQSITGNWVAWYQQRPGKAPRLLIYRGAALLGGVPSRFSGSAAGTDFTLTIGNLQTEDFGTFYCQQYDTYPGTFG QGTKVEVKRTVAAPSVF;and (3630LC; SEQ ID NO: 33)DIQMTQSPSTLSASIGDTVRISCRASQSITGGWLAWYHQRPGKAPRLLIYRGSRLLGGVPSRFSGSAAGTDFTLTIANLQAEDFGTFYCQQYDTYPGTFG QGTKVEVKRTVAAPSVF.

Accordingly, one aspect of this invention features an isolatedpolypeptide comprising the sequence of any one of SEQ ID NOs: 1-33. Theinvention also provides an isolated anti-HIV antibody comprising one orboth of a heavy chain comprising the sequence of any one of SEQ ID NOs:1-18 and a light chain comprising the sequence any one of SEQ ID NOs:19-33.

The above-mentioned antibody can be a human antibody, a chimericantibody, or a humanized antibody. It can be an IgG1, IgG2, IgG3, orIgG4. The antibodies of the invention recognize the epitope on theenvelope spike recognized by 8ANC1.95 and are broadly neutralizing.

In another aspect, the invention provides an isolated nucleic acidencoding the isolated polypeptide or anti-HIV-1 antibody describedabove. Also provided are a vector comprising the nucleic acid and acultured cell comprising the nucleic acid.

In another aspect, the invention provides a composition comprising atleast one of the above-described isolated polypeptide or anti-HIV-1antibody or a fragment thereof. In one embodiment, the compositioncomprises a pharmaceutically acceptable carrier.

In another aspect, the invention provides a method of preventing ortreating an HIV-1 infection or an HIV-related disease. The methodincludes steps of identifying a patient in need of such prevention ortreatment, and administering to the patient a first therapeutic agentcomprising a therapeutically effective amount of at least one of theabove-described isolated polypeptide or arid-HIV-1 antibody. The methodcan further comprise administering a second therapeutic agent, such asan antiviral agent.

In another embodiment, the present invention provides an isolatedantigen comprising an epitope-scaffold that mimics the HIV-1 envelopespike epitope of broadly neutralizing antibody 8ANC195. In one aspect,the epitope-scaffold comprises a discontinous epitope and a scaffold. Inanother aspect, the epitope is derived from HIV-1 gp120 and gp41, and atleast part of the scaffold is not derived from gp120 or gp41_(—) Inanother apect, the discontinuous epitope comprises amino acidscorresponding to amino acid numbers 44-47, 90-94, 97, 234, 236-238, 240,274-278, 352-354, 357, 456, 463, 466, 487, and 625-641 of gp140 from HIVstrain 93TH057 numbered using standard numbering for HIV strain HXBC2.The amino acids corresponding to amino acid numbers 234 and 276 may beglycosylated.

In another aspect, the invention provides an isolated nucleic acidencoding the isolated antigen described above. Also provided are avector comprising the nucleic acid and a cultured cell comprising thenucleic acid.

In another aspect, the invention provides a composition comprising theisolated antigen. In one embodiment, the composition further comprises apharmaceutically acceptable carrier, in one embodiment, the compositionfurther comprises an adjuvant, in another aspect, the present inventionprovides a method for generating an immune response in a subject in needthereof, comprising administering to said subject a compositioncomprising the above-described isolated antigen in an amount effectiveto generate an immune response.

In another aspect, the invention provides a method of preventing ortreating an HIV-1 infection or an HIV-related disease. The methodincludes steps of identifying a patient in need of such prevention ortreatment, and administering to the patient a first therapeutic agentcomprising a therapeutically effective amount of the above-describedantigen. The method can further comprise administering a secondtherapeutic agent, such as an antiviral agent.

In another aspect, the present invention provides a method for detectingor isolating an HIV-1 binding antibody in a subject comprising obtaininga biological sample from the subject, contacting the sample with theabove-described antigen, and conducting an assay to detect or isolate anHIV-1 binding antibody.

The details of one or more embodiments of the invention are set forth inthe description below. Other features, objects, and advantages of theinvention will be apparent from the description and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and B illustrates alignments of (A) VH and (B) VL sequences ofmature 8ANC195 and its putative germ-line progenitor (GI.). 8ANC195 HChas the sequence of SEQ ID NO: 34. 8ANC195 LC has the sequence of SEQ IDNO: 35. GL has the sequence of SEQ ID NO: 36. GL LC has the sequence ofSEQ ID NO: 37. Residues forming the CDR loops are labeled (CDR 1),(CDR2) and (CDR3). 8ANC195 is one of the most heavily mutated bhAbsisolated to date, with 49 of 103 amino acid mutations in the ITC and 25of 90 in the LC. The HC was too highly somatically mutated to accuratelyassign D and J gene segments, but the LC showed sufficient homology toassign its J segment as IGKJ5*01.

FIGS. 2A-C illustrates conformations of 8ANC195 CDRH1 and CDRH3 loops.(A) The hook-like conformation of CDRH1 is stabilized by burial of thehydrophobic Phe30HC side chain and hydrogen bonds within CDRH1 and withCDRH3 and FWR1 and FWR3 residues (Ala24HC and Asp73HC, respectively).(B) The complexed. CDRH3 conformation consists of a protruding loop(residues 95HC-100HC) and a small β-sheet subdomain (residues 100dHC-100 kHC:) stabilized by multiple hydrogen bonds within CDRH3 as wellas with CDRH1 and CDRL3. A hydrogen bond between Tyr92LC and Gly100cHCstabilized the bifurcation of CDRH3 into its two subdomains. CDRH1 andCDRH3 loop backbone atoms are shown as sticks and side chains ofresidues important for stabilizing, the loop conformations are shown assticks (involved in direct contacts) or lines (backbone involved incontacts) (other side chains Tyr98HC, Lys100HC and Trp100aHC, are shownfor clarity). (C) comparison of CDRH3 loops in 8 ANC195 and otheranti-HIV-1 bNAbs. CDRH3 residues corresponding to 8ANC195HC residues90-105 of NIH45-46 (PDB 3U7 Y), PG16 (PDB 4DQO), PGT121 (PDB 4FQC) andPGT-128 (PDB 3TYG) are shown as Cα traces.

FIGS. 3A-E illustrates CD4 interactions with 8ANC195. (A)Superimposition of sCD4 DID2/gp120 structures (ribbon diagrams) fromcomplexes with 8ANC195, 17b (PDB 1GCI) and 21c PDB 3LQA). (B)Competition ELISA of 8ANC195 IgG binding to 93TH057 gp120 in thepresence of increasing concentrations of potential competitors (sCD4,diamonds; J3 VHH, triangles; 3BNC60 Fab, squares; NIH45-46 Fab,circles). No competition was observed with small, single-Ig domainCD4-binding site ligands (sCD4, J3 VHH), but larger Fab fragments of CD4binding site antibodies (3BNC60, NIH45-46) competed for binding. (C) Invitro assay comparing neutralization of YU2 by sCD4 (squares), 8ANC195IgG (triangles), and an equimolar mixture of 8ANC195 and sCD4 (circles).(D) Packing of 8ANC195/sCD4/gp120 crystals. Several symmetry mates areshown as surface representations (8ANC195HC; 8ANC195 LC; 93TH057 gp120;sCD4 D1D2). Areas where two complexes form crystal contacts areindicated. (E) In vitro assay comparing neutralization of YU2 by 8ANC195IgG (squares), 3BNC60 IgG (triangles), and an 8ANC195 IgG mutant thatlacks the FWR3 insertion (Ser77a-Pro77b-Pro77c-Ile77d) that results inthe protruding “FWR3_(HC)thumb” (circles).

FIGS. 4A-I illustrates surface area buried at interface of 8ANC195 Faband gp120. Left panels, surface area buried on 8ANC195 Fab by (A) gp120protein, residues, (C) Asn276gp120 glycan or (E) Asn234gp120 glycan;right panels, surface area buried by 8ANC195 Fab on (B) gp120 proteinresidues, (D) Asn234gp120 glycan or (F) Asn276gp120 glycan. Atoms buriedat these interfaces are shown as surface representations overlaid ontoribbon diagrams of 8ANC195 Fab and gp120 or stick representations ofglycans. 8ANC195 Fab: HC; LC; CDRH1; CDRH2; CDRH3: gp120: inner domain;outer domain; loop D; loop V5; Asn234gp120 glycan: Asn276gp120 glycan.2Fo-Fe annealed omit electron density maps (grey mesh, o=1) used tobuild (E) Asn234_(gp120) glycan and (H) Asn276_(gp120) glycan. (1)Modeled fucose residue β1-6-linked to the first N-acetylglucosamineresidue of the Asn276_(gp120) glycan shows that the core fucose of acomplex-type N-glycan could be accommodated by the 8ANC195. Glycanresidues are shown as sticks, and gp120, 8ANC195 HC and CD4 are shown assurface representations.

FIGS. 5A and B illustrates a comparison of glycan-dependent bNAbs.8ANC195 is “bracketed” by two glycans (Ash234_(gp120) glycan;Asn276_(gp120) glycan) in the 8ANC195 Fab/gp120/sCD4 complex structure(left panels). For comparison, crystal structures of PG16 (middlepanels, PDB 4DQO) bound to a V1/V2 loop scaffold and PGT128 (rightpanels, PDB 3TYG) bound to a V3 loop scaffold are shown with (A) theantibody HCs aligned to the 8ANC195 HC or (B) an alternative viewshowing their interactions with bracketing glycans (for PG16:Asn160_(gp120) glycan/Asn 172_(gp120) glycan; for PGT128: Asn301_(gp120)glycan/Asn332_(gp120) glycan). The proteins are shown as ribbon diagramsand the glycans as stick representations.

FIGS. 6A-C illustrate green EM refinement statistics. (A) Electronmicrograph at 52,000× magnification and −0.8 μm defocus. (B)Reference-free 2D class averages of the SOSIP trimer in complex with8ANC195 Fab showing various orientations. (C) Fourier Shell Correlation(FSC) graph resulting from refinement. The resolution was determined as18.7 Å at an PSC cut-off of 0.5.

FIGS. 7A. and B illustrates negative stain EM reconstruction of BG505SOSIP.664 in complex with 8ANC195 Fab fit two ways. (A) When thegp120-8ANC195 Fab structure was fit into the EM density, the gp120 fromthe complex structure was displaced slightly outwards in comparison tothe gp120 in the SOSIP trimer structure. The HC and LC of the Fab areshown. The Asn234_(gp120) and Asn276_(gp120) glycans are shown asspheres. (B) Close up of the Fab-Env interface. The position ofAsn637_(gp120) can be deduced from the position of the C-tenninus ofHR2, which corresponds to residue Gly664 ₄₁. This residue is in closeproximity to the LC and the glycan at this position could interact withthe 8ANC195 Fab.

FIGS. 8A-D illustrates EM reconstruction of 8ANC195 FabBG505 SOSIP.664showing gp41 contacts. Top view of EM density with the X-ray structuresof BG505 SOSIP.664 (PDB ID 4NCO; gp120, grey; gp41) and 8ANC195 Fab (HC;LC with a map contour level of 0.0176 (A) and 0.030 (B). Areas ofcontact between 8ANC195 and gp41 are marked with circles, those between8ANC195 and gp120 with black circles. (C,D) Close-up of 8ANC195 LC andHR2 region in EM complex structure (HR2 coordinates in PDB 4NCO withpresumptive sidechains for strain YU2 added to the polyalaninecoordinates). (C) Fab is shown as a surface representation withhighlights (CDRL1; CDRL2; CDRH3, and gp41 HR2 is shown as a ribbondiagram. The position of Asn637gp41 was deduced from the position of theC-terminus of the SOSIP.664 trimer (Gly664gp41). (D) 8ANC195 HC and LCresidues (sticks) positioned to contact HR2, with side chains ofsurface-exposed residues that vary between newly isolated 8ANC195variants shown as sticks.

FIGS. 9A-C relate to Single Cell Variants of 8ANC195. (A) Strategy oflarge scale single cell sorting. (B) IgH and IgL chain genes fromisolated single cell variants of 8ANC195. Identical members are groupedtogether. The HC CDR3 of 8ANC195 has the sequence of SEQ ID NO: 38. TheHC CDR3 of 8ANC142 has the sequence of SEQ ID NO: 39. The HC CDR3 of8ANC3430 has the sequence of SEQ ID NO: 40. The HC CDR3 of 8ANC3484 hasthe sequence of SEQ ID NO: 41. The HC CDR3 of 8ANC3044 has the sequenceof SEQ ID NO: 42 The HC CDR3 of 8ANC3630 has the sequence of SEQ ID NO:43. The LC CDR3 of 8ANC195 has the sequence of SEQ ID NO: 44. The LCCDR3 of 8ANC142 has the sequence of SEQ ID NO: 45. The LC CDR3 of8ANC3430 has the sequence of SEQ ID NO: 46. The LC CDR3 of 8ANC3484 hasthe sequence of SEQ ID NO: 47. The LC CDR3 or 8ANC3044 has the sequenceof SEQ ID NO: 48. The LC CDR3 of 8ANC3630 has the sequence of SEQ ID NO:49. (C) IC₅₀ neutralization titers of distinct single cell versions ofthe 8ANC195 clone compared to 8ANC195 against a 15 virus Tier 2 panel.

FIGS. 10A and B depict the alignment of amino acid sequences of alldistinct single cell versions of the 8ANC195 clone. HC (A) and LC (B)sequences were aligned with the respective germline genes. Mutationsintroduced by somatic hypermutation are indicated.

FIGS. 11A-C are directed to Bulk Sorted Variants of 8ANC195. (A)Strategy of bulk memory B cell sorting without antigen. (B) PCR strategyfor the amplification of 8ANC195 HC and LC clone members. Shown are thepriming sites aligned with the original nucleotide sequence of 8ANC195at the respective sites. Mismatches with the respective germline genesare indicated. Primers 1 and 2 for the 8ANC195 HC FWR1 have SEQ ID Nos:50 and 51, respectively. Primers 1 and 2 for the 8ANC195 LC FWR1 haveSEQ ID Nos: 52 and 53, respectively. The primer for the 8ANC195 BCJ-gene has SEQ ID NO: 54. Primers 1 and 2 for the 8ANC195 LC J-gene haveSEQ ID Nos: 55 and 56, respectively. (C) Phylogenetic tree of 128isolated BC and 100 LC sequences. Representative members chosen foralignment are indicated.

FIGS. 12A and B depict alignment of amino acid sequences of selectedbulk sorted versions of the 8ANC195 clone. HC (A) and LC (B) sequenceswere aligned with the respective germline genes as well as the original8ANC195 sequence. All mutations introduced by somatic hypermutation areindicated.

FIGS. 13A and B show that δ52 _(EC)κ5_(EC) is more potent than8ANC195IC₅₀ values of δ52HC κ51C and 8ANC195 against Tier 2 15 viruspanel shown as dot plot (A) and Table (B). NT, not tested.

FIGS. 14A-C show that somatic mutations in the 8ANC195 LC CDRs and FWRscould affect contacts with gp41. (A) Surface representation of 8ANC195Fab (HC; LC; somatically mutated, surface-exposed LC residues; residue64_(LC)). (B) Surface representation of 8ANC195 Fab and BG505 gp41 HR2with a modeled Man6 sugar attached to Asn637gp41, (C) Surfacerepresentation of 8ANC195 Fab (CDRL1; CDRL2; CDRH3; residue (64_(LC))and BG505 gp41 HR2 with a modeled Man6 sugar attached to Asn637gp41.

FIG. 15 illustrates locations of bNAb epitopes tin HIV-1 Env Trimer. EMdensity map of Env trimer including MPER region showing approximateepitope locations for antibodies targeting the 8ANC195 epitope CD4binding site, V3 loop/Asn332 glycan (332 glycan shown as spheres), V1/V2loop/Asn160 glycan (160 glycan shown as spheres), and MPER

FIGS. 16A and C illustrate crystal structures of 8ANC195 Fab and8ANC195/Qp120/sCD4 complex. (A) Superimposition of unbound and bound (HCand LC) structures of 8ANC195 Fab shown as ribbon diagrams. CDR loopsare highlighted (CDRH1/CDRL1; CDRH21CDRL2; CDRH3; CDRL3) and a“thumb”-like loop formed by an insertion in FWR3 is indicated.Disordered loops are shown as dashed lines. (B) Space-filling model(inset) and ribbon diagram of ternary complex of 8ANC195 (HC and LC),sCD4, and 93TH057 gp120 core (inner domain; outer domain; bridgingsheet; loop D; loop V5; CD4 binding loop). Ordered glycans attached toAsn234_(gp120) and Asn276_(gp120) are shown as sticks. Fab CDR loops areindicated as in (A), sCD4 was omitted from the right panel for clarity.(C) Approximate locations of bNAb epitopes on a surface representationof the gp120 core. The epitopes of V3 and V1/V2 antibodies includeregions of loops (dotted lines) not present in the gp120 core structure.CD4 binding site and 8ANC195 epitopes are outlined by black (CD4 bindingsite) and (8ANC195) dots. Glycans included in the 8ANC195 epitope areindicated. Subdomains of gp120 are indicated as in (B).

FIGS. 17A -E show contacts made by 8ANC195 HC with gp120 proteinresidues and glycans. Labels for gp120 protein and glycan residues arcitalicized. Hydrogen bonds are shown as dashed lines. (A) FWR3_(BC) loopcontacts with loop D, loop V5, and outer domain loop. (B) 8ANC195 HCCDRH1 and CDRH3 contacts with gp120 inner domain. (C) Buried surfacearea between the Asn234_(gp120) glycan (transparent surfilee with glycanresidues shown as sticks) and 8ANC195 (HC FWR residues and CDRH2 areindicated). Antibody atoms buried by glycan interactions are shown assurfaces. (0) Buried surface area between the Asn276 glycan_(gp120)(transparent surface with glycan residues shown as sticks) and 8ANC195(HC FWR residues and CDRH1 are indicated). Antibody atoms buried byglycan interactions are shown as surfaces. (E) Top: Contacts made by8ANC195 HC FWR residues and CDRH2 with Asn234_(gp120) glycan. Glycan andprotein residues involved in hydrogen bonds are shown as sticks. Bottom:schematic of ordered high mannose glycans on Asn234_(gp120) andAsn276_(gp120) (bottom).

FIGS. 18A and 13 show the EM structure of 8ANC195/Env trimer complex andmodel of 8ANC195 LC interactions with gp41 HR2. (A) EM reconstruction of8ANC195 Fab/BG505 SOSIP.664. Side (left) and top (right) views of EMdensity with the X-ray structures of BG505 SOSIP.664 (PDB ID 4NCO;gp120, gp41) and 8ANC195 Fab fit in two ways: (i) fitting 8ANC195 Fabindependently of gp140 coordinates to the EM density (bestfit/independently placed), and (ii) by aligning the gp120 of thegp120/8ANC195 complex structure onto the gp120 of PDB 4NCO fit to the EMdensity. (B) Close-up of 8ANC195 LC/HR2 region of EM complex structure(Fab placement is best fit, independently placed as in (A)). Left: Fabis shown as a surface representation with highlights (CDRL1: CDRL2;CDRH1; CDRH3), and gp140 is shown as a ribbon diagram (gp120; gp41). Theposition of Asn637_(gp41) was deduced from the position of theC-terminus of the SOSIP.664 trimer (Gly664_(gp41)). Right: 8ANC195 HCand LC residues (sticks) positioned to contact HR2, which is shown as asurface representation calculated trona HR2 coordinates in PDB 4NCO withpresumptive sidechains added to the polyalanine coordinates.

FIGS. 19A-C show effects of LC sequence changes on 8ANC195neutralization potency. (A) Sequences of LC CDRs in constructs used with8ANC195 HC to make chimeric IgGs (left) and location of CDRs on 8ANC195structure (right). Sequences derived from the mature antibody are shownand those derived from the germline precursor are shown on a greybackground. The mutations introduced into CDRL3 in gICDRL3Ala are shownon a white background. (B) Effects of changes in 8ANC195 LC on bindingto 93TH057 and YU2 gp120s and neutralization of viral strains, expressedas fold changes over results for 8ANC195 IgG, ICD and IC₅₀ values forthese experiments are shown in table S3, (C) Heat map showing theexpression and neutralization of randomly paired HCs and LCs from thebulk sort on a Tier 2 15-virus panel. Average IC₅₀ values (arithmeticmeans) between 0.1 and 2 μg/ml; between 2.1 and 10 μg/ml, between 10.1and 14.9 μg/ml, and above 15 μg/ml are indicated with varying degrees ofshaded squares. Empty squares represent insufficient antibodyexpression.

FIG. 20 depicts the alignment of gp140 sequences from HIV strains HXBC2and 93TH057 using standard HXBC2 numbering of amino acid residues. Aminoacid residues contacted by 8ANC195 in the complex crystal structure with93TH057 gp120 core are indicated on the 93TH057 sequence. Glycanscontacted by 8ANC195 in the complex crystal structure with 93TH057 gp120core are shown as the asparagine residues to which they are attached,highlighted in cyan on the 93TH057 sequence. The region of gp41contacted by 8ANC195 based on the EM complex structure is indicated.Select glycans are shown as diagrams on the asparagine residues to whichthey are attached.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based, at least in part, on the identificationof the epitope recognized by 8ANC195, a broadly neutralizing antibody tothe HIV-1 envelope glycoprotein. The present invention, in oneembodiment, provides an isolated antigen comprising the epitope,compositions comprising the antigen, and methods of using the antigen.In other embodiments, the present invention provides isolated ant-HIV-1antibodies that recognize the epitope on the HIV-1 envelope spikerecognized by 8ANC195, compositions comprising the antibodies, andmethods of using the antibodies.

In one embodiment, the present invention is directed to an isolatedanti-HIV antibody comprising one or both of a heavy chain comprising thesequence of any one of SEQ ID NOs: 1-18 and a light chain comprising thesequence any one of of SEQ ID NOs: 19-33. In one preferred embodiment,the heavy chain comprises the sequence of SEQ ID NO: 1 and the lightchain comprises the sequence of SEQ ID NO: 20. In another embodiment,the present invention provides an d polypeptide comprising the sequenceof any one of SEQ ID NOs: 1-33.

The above-mentioned antibody can be a human antibody, a chimericantibody, or a humanized antibody. It can be an. IgG1, IgG2. IgG3, orIgG4. The antibodies of the invention recognize the epitope on the HIV-1envelope spike recognized by 8ANC195 and are broadly neutralizing.8ANG195 is known in the art and disclosed, for example, by Scheid etal., Science, 333, 1633 (2011). The heavy chain of 8ANC195 has thesequence of SEQ ID NO: 34 and the light chain of 8ANC195 has thesequence of SEQ ID NO: 35.

The term “antibody” (Ab) as used herein includes monoclonal antibodies,polyclonal antibodies, multispecific antibodies (for example, bispecificantibodies and polymactive antibodies), and antibody fragments. Thus,the term “antibody” as used in any context within this specification ismeant to include, but not be limited to, any specific binding member,immunoglobulin class and/or isotype (e.g., IgG 1, IgG2, IgG3, IgG4, IgM,IgA, IgD IgE and IgM); and biologically relevant fragment or specificbinding member thereof, including but not limited to Fab, F(ab′)2, Fv,and scFv (single chain or related entity). It is understood in the artthat an antibody is a glycoprotein comprising at least two heavy (H)chains and two light (L) chains inter-connected by disulfide bonds, oran antigen binding portion thereof. A heavy chain is comprised of aheavy chain variable region (VH) and a heavy chain constant region (CH1,CH2 and CH3). A light chain is comprised of a light chain variableregion (VI) and a light chain constant region (CL). The variable regionsof both the heavy and light chains comprise framework regions (FWR) andcomplementarity determining regions (CDR). The four EWR regions arerelatively conserved while CDR regions (CDR1, CDR2 and CDR3) representhypervariable regions and are arranged from NH2 terminus to the COOHterminus as follows: FWR1, CDR1, FWR2, CDR2, FWR3, CDR3, and FWR4. Thevariable regions of the heavy and light chains contain a binding domainthat interacts with an antigen while, depending of the isotype, theconstant region (s) may mediate the binding of the immunoglobulin tohost tissues or factors.

Also included in the definition of “antibody” as used herein arechimeric antibodies, humanized antibodies, and recombinant antibodies,human antibodies generated from a transgenic non-human animal, as wellas antibodies selected from libraries using enrichment technologiesavailable to the artisan.

The term “variable” refers to the fact that certain segments of thevariable (V) domains differ extensively in sequence among antibodies.The V domain mediates antigen binding and defines specificity of aparticular antibody for its particular antigen. However, the variabilityis not evenly distributed across the 110-amino acid span of the variableregions. Instead, the V regions consist of relatively invariantstretches called framework regions (FRs) of 15-30 amino acids separatedby shorter regions of extreme variability called “hypervariable regions”that are each 9-12 amino acids long. The variable regions of nativeheavy and light chains each comprise four FRs, largely adopting a betasheet configuration, connected by three hypervariable regions, whichform loops connecting, and in some cases forming part of, the beta sheetstructure. The hypervariable regions in each chain are held together inclose proximity by the FRs and, with the hypervariable regions from theother chain, contribute to the formation of the antigen-binding site ofantibodies (see, for example, Kabat et al., Sequences of Proteins ofImmunological Interest, 5th Ed. Public. Health Service, NationalInstitutes of Health, Bethesda, Md. (1991)).

The term “hypervariable region” as used herein refers to the amino acidresidues of an antibody that are responsible for antigen binding. Thehypervariable region generally comprises amino acid residues from a“complementarity determining region” (“CDR”).

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. The term “polyclonal antibody” refers to preparationsthat include different antibodies directed against differentdeterminants (“epitopes”).

The monoclonal antibodies herein include “chimeric” antibodies in whicha portion of the heavy and/or light chain is identical with, orhomologous to, corresponding sequences in antibodies derived from aparticular species or belonging to a particular antibody class orsubclass, while the remainder of the chain(s) is identical with, orhomologous to, corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired biological activity (see, for example, U.S. Pat. No. 4,816,567;and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)).Chimeric antibodies include antibodies having one or more human antigenbinding sequences (for example, CDRs) and containing one or moresequences derived from a non-human antibody, for example, an FR or Cregion sequence. In addition, chimeric antibodies included herein arethose comprising a human variable region antigen binding sequence of oneantibody class or subclass and another sequence, for example. FR or Cregion sequence, derived from another antibody class or subclass.

A “humanized antibody” generally is considered to be a human antibodythat has one or more amino acid residues introduced into it from asource that is non-human. These non-human amino acid residues often aare referred to as “import” residues, which typically are taken from an“import” variable region. Humanization may be performed following themethod of Winter and co-workers (see, for example, Jones et at., Nature121:522-525 (1986); Reichmann et al., Nature 332:323-327 (1988);Verhoeyen et al., Science 239:1534-1536 (1988)), by substituting importhypervariable region sequences for the corresponding sequences of ahuman antibody. Accordingly, such “humanized” antibodies are chimericantibodies (see, for example, U.S. Pat. No. 4816,567), wheresubstantially less than an intact human variable region has beensubstituted by the corresponding sequence from a non-human species.

An “antibody fragment” comprises a portion of an intact antibody, suchas the antigen binding or variable region of the intact antibody.Examples of antibody fragments include, but are not limited to, Fab,Fab′, F(ab)2, and Fv fragments; diabodies; linear antibodies (see, forexample, U.S. Pat. No. 5,641,870; Zapata et al., Protein Eng. 8(10):1057-1062 [1995]); single-chain antibody molecules; and multispecificantibodies formed from antibody fragments.

“Tv” is the minimum antibody fragment that contains a completeantigen-recognition and antigen-binding site. This fragment contains adimer of one heavy- and one light-chain variable region domain in tight,non-covalent association. From the folding of these two domains emanatesix hypervariable loops (three loops each from the H and L chain) thatcontribute the amino acid residues for antigen binding and conferantigen binding specificity to the antibody. However, even a singlevariable region (or half of an Fv comprising only three CDRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

“Single-chain Fv” (“sFv” or “seFv”) are antibody fragments that comprisethe VH and VL antibody domains connected into a single polypeptidechain. The sFv polypeptide can further comprise a polypeptide linkerbetween the VH and VL domains that enables the sFv to form the desiredstructure for antigen binding. For a review of sFv, see, for example,Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113,Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994);Borrebaeck 1995, infra.

The term “diabodies” refers to small antibody fragments prepared byconstructing sFv fragments with short linkers (about. 5-10 residues)between the VH and VL domains such that inter-chain but not intra-chainpairing of the V domains is achieved, resulting in a bivalent fragment,i.e., fragment having two antigen-binding sites. Bispecific diabodiesare heterotrimer of two “crossover” sFv fragments in which the VH and VLdomains of the two antibodies are present on different polypeptidechains. Diabodies are described more fully in, for example, EP 404,097;WO 93/11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA,90:6444-6448 (1993).

Domain antibodies (dAbs), which can be produced in fully human form, arethe smallest known antigen-binding fragments of antibodies, ranging fromabout 11 kDa to about 15 kDa. DAbs are the robust variable regions ofthe heavy and light chains of immunoglobulins (VH and VL, respectively).They are highly expressed in microbial cell culture, show favorablebiophysical properties including, for example, but not limited to,solubility and temperature stability, and are well suited to selectionand affinity maturation by in vitro selection systems such as, forexample, phage display. DAbs are bioactive as monomers and, owing totheir small size and inherent stability, can be formatted into largermolecules to create drugs with prolonged serum half-lives or otherpharmacological activities. Examples of this technology have beendescribed in, for example, WO9425591 for antibodies derived fromCamelidae heavy chain as well in US20030130496 describing the isolationof single domain fully human antibodies from phage libraries.

Fv and sFv are the only species with intact combining sites that aredevoid of constant regions. Thus, they are suitable for reducednonspecific binding during in vivo use. sFv fusion proteins can beconstructed to yield fusion of an effector protein at either the aminoor the carboxy terminus of an sFv. See, for example, AntibodyEngineering, ed. Borrebaeck, supra. The antibody fragment also can be a“linear antibody”, for example, as described in U.S. Pat. No. 5,641,870for example. Such linear antibody fragments can be monospecific orbispecific.

In certain embodiments, antibodies of the described invention arebispecific or multi-specific. Bispecific antibodies are antibodies thathave binding specificities for at least two different epitopes.Exemplary bispecific antibodies can bind to two different epitopes of asingle antigen. Other such antibodies can combine a first antigenbinding site with a binding site for a second antigen. Alternatively, ananti-HIV arm can be combined with an arm that binds to a triggeringmolecule on a leukocyte, such as a T-cell receptor molecule (forexample, CD3), or Fe receptors for IgG (Fe gamma R), such as Fr gamma RI(CD64), Fe gamma RII (CD32) and Fe gamma RIII (CD16), so as to focus andlocalize cellular defense mechanisms to the infected cell. Bispecificantibodies also can be used to localize cytotoxic agents to infectedcells. Bispecific antibodies can be prepared as full length antibodiesor antibody fragments (for example, F(ab′)2 bispecific antibodies). Forexample, WO 96/16673 describes a bispecific anti-ErbB2/anti-Fc gammaRIII antibody and U.S. Pat. No. 5,837,234 discloses a bispecificanti-ErbB2/anti-Fc gamma RI antibody. For example, a bispecificanti-ErbB2/Fc alpha antibody is reported in WO98102463; U.S. Pat. No.5,821,337 teaches a bispecific anti-ErbB2/anti-CD3 antibody. See also,for example, Mouquet et al., Polyreactivity increases The ApparentAffinity Of Anti-HIV Antibodies By Heteroligation, NATURE. 467, 591-5(2010).

Methods for making bispecific antibodies are known in the art.Traditional production of full length bispecific antibodies is based onthe co-expression of two immunoglobulin heavy chain-light chain pairs,where the two chains have different specificities (see, trir example,Millstein et al., Nature, 305:537-539 (1983)). Similar procedures aredisclosed in, for example, WO 93/08829, Trannecker et al., EMBO J.,10:3655-3659 (1991) and see also; Mouquet et al., PolyreactivityIncreases The Apparent Affinity Of Anti-HIV Antibodies ByHeteroligation, NATURE, 467, 591-5 (2010).

Alternatively, antibody variable regions with the desired bindingspecificities (antibody-antigen combining sites) are fused toimmunoglobulin constant domain sequences. The fusion is with an Ig heavychain constant domain, comprising at least part of the hinge, CH2, andCH3 regions. According to some embodiments, the first heavy-chainconstant region (CH1)containing the site necessary for light chainbonding, is present in at least one of the fusions. DNAs encoding theimmunoglobulin heavy chain fusions and, if desired, the immunoglobulinlight chain, are inserted into separate expression vectors, and areco-transfected into a suitable host cell. This provides for greaterflexibility in adjusting the mutual proportions of the three polypeptidefragments in embodiments when unequal ratios of the three polypeptidechains used in the construction provide the optimum yield of the desiredbispecific antibody. It is, however, possible to insert the codingsequences for two or all three polypeptide chains into a singleexpression vector when the expression of at least two polypeptide chainsin equal ratios results in high yields or when the ratios have nosignificant affect on the yield of the desired chain combination.

Techniques for generating bispecific antibodies from antibody fragmentsalso have been described in the literature. For example, bispecifieantibodies can be prepared using chemical linkage. For example, Brennanet al., Science, 229: 81 (1985) describe a procedure wherein intactantibodies are proteolytically cleaved to generate F(ab′)2 fragments.These liagments are reduced in the presence of the dithiol complexingagent, sodium arsenite, to stabilize vicinal dithiols and preventintermolecular disulfide formation. The Fab′ fragments generated thenare converted to thionitrobenzoate (TNB) derivatives. One of theFab′-TNB derivatives then is reconverted to the Fab′-thiol by reductionwith mercaptoethylamine and is mixed with an equimolar amount of theother Fab′-TNB derivative to form the bispecifie antibody. Thebispecifie antibodies produced can be used as agents for the selectiveimmobilization of enzymes.

Other modifications of the antibody are contemplated herein. Forexample, the antibody can be linked to one of a variety ofnonproteinaceous polymers, for example, polyethylene glycol,polypropylene glycol, polyoxyalkylenes, or copolymers of polyethyleneglycol and polypropylene glycol. The antibody also can be entrapped inmicrocapsules prepared, example, by cooperation techniques or byinterfacial polymerization (For example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate)microcapsules,respectively), in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules), or in macroemulsions. Such techniques are disclosed in,for example, Remington's Pharmaceutical Sciences, 16th edition, Oslo,A., Ed., (1980).

Typically, the antibodies of the described invention are producedrecombinantly, using vectors and methods available in the art. Humanantibodies also can be generated by in vitro activated B cells (see, forexample, U.S. Pat. Nos. 5,567,610 and 5,229,275). General methods inmolecular genetics and genetic engineering useful in the presentinvention are described in the current editions of Molecular Cloning: ALaboratory Manual (Sambrook, et al., 1989, Cold Spring Harbor LaboratoryPress), Gene Expression Technology (Methods in Enzymology, Vol. 185,edited by D. Goeddel., 1991, Academic Press, San Diego, Calif.), “Guideto Protein Purification” in Methods in. Enzymology (M. P. Deutshcer,ed., (1990) Academic Press, Inc.); PCR Protocols: A Guide to Methods andApplications (Innis, et at. 1990. Academic Press, San Diego, Calif.),Culture of Animal Cells: A Manual of Basic Technique, 2nd Ed. (R. I.Freshney. 1987. Liss, Inc. New York, N.Y.), and Gene Transfer andExpression Protocols, pp. 109-128, ed. E. J. Murray. The Humana PressInc., Clifton, N.J.). Reagents, cloning vectors, and kits for geneticmanipulation are available from commercial vendors such as BioRad,Stratagem, Invitrogen, ClonTech and Sigma-Aldrich Co.

Human antibodies also can be produced in transgenic animals (forexample, mice) that are capable of producing a full repertoire of humanantibodies in the absence of endogenous immunoglobulin production. Forexample, it has been described that the homologous deletion of theantibody heavy-chain joining region (JH) gene in chimeric and germ-linemutant mice results in complete inhibition of endogenous antibodyproduction. Transfer of the human germ-line immunoglobulin gene arrayinto such germ-line mutant mice results in the production of humanantibodies upon antigen challenge. See, for example, Jakobovits et al.,Proc. Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits et al., Nature.362:255-258 (1993); Bruggemann et al., Year in Immuno., 7:33 (1993);U.S. Pat. Nos. 5,545,806, 5,569,825, 5,591,669 (all of GenPharm.); Pat.No. 5,545,807; and WO 97/17852. Such animals can be geneticallyengineered to produce man antibodies comprising a polypeptide of thedescribed invention.

Various techniques have been developed for the production of antibodyfragments. Traditionally, these fragments were derived via proteolyticdigestion of intact antibodies (see, for example, Morimoto et al.,Journal of Biochemical and Biophysical Methods 24:107-117 (1992); andBrennan et al., Science, 229:81 (1985)). However these fragments can nowbe produced directly by recombinant host cells. Fab, Fv and ScFvantibody fragments can all be expressed in and secreted from E. coli,thus allowing the facile production of large amounts of these fragments,Fab′-SH fragments can be directly recovered from E. coli and chemicallycoupled to form F(ab′)2 fragments (see, for example, Carter et al.,Bio/Technology 10:163467 (1992)). According to another approach. F(ab′)2fragments can be isolated directly from recombinant host cell culture.Fab and F(ab′)2 fragment with increased in vivo half-life comprising asalvage receptor binding epitope residues are described in U.S. Pat, No,5,869,046. Other techniques for the production of antibody fragmentswill be apparent to the skilled practitioner.

Other techniques that are known in the art for the selection of antibodyfragments from libraries using enrichment technologies, including butnot limited to phage display, ribosome display (Hanes and Pluckthun,1997, Proc. Nat. Acad. Sci. 94: 4937-L1942), bacterial display(Georgiou, et al., 1997, Nature Biotechnology 15: 29-34) and/or yeastdisplay (Kieke, et al., 1997, Protein Engineering 10: 1303-1310) may beutilized as alternatives to previously discussed technologies to selectsingle chain antibodies. Single-chain antibodies are selected from alibrary of single chain antibodies produced directly utilizingfilamentous phage technology. Phage display technology is known in theart (e.g., see technology from Cambridge Antibody Technology (CAT)) asdisclosed in U.S. Pat. Nos. 5,565,332; 5,733,743; 5,871,907; 5,872,215,5,885,793; 5,962,255, 6,140,471; 6,225,447; 6,291650; 6,492,160;6,521,404; 6.544,731; 6,555,313; 6,582,915; 6,593, 081, as well as otherU.S. family members, or applications Which rely on priority filing GB9206318, filed 24 May 1992; see also Vaughn, et al, 1996, NatureBiotechnology 14: 309-314). Single chain antibodies may also be designedand constructed using available recombinant DNA technology, such as aDNA amplification method (e.g., PCR.), or possibly by using a respectivehybridoma cDNA as a template.

Variant antibodies also are included within the scope of the invention.Thus, variants of the sequences recited in the application also areincluded within the scope of the invention. Further variants of theantibody sequences having improved affinity can be obtained usingmethods known in the art and are included within the scope of theinvention. For example, amino acid substitutions can be used to obtainantibodies with further improved affinity. Alternatively, codonoptimization of the nucleotide sequence can be used to improve theefficiency of translation in expression systems for the production ofthe antibody.

The present invention provides for antibodies., either alone or incombination with other antibodies, such as, but not limited to, VRC01,anti-V3 loop, CD4bs, and CD4i antibodies as well as PG9/PG16-likeantibodies, that have broad neutralizing activity in serum.

The present invention also relates to isolated polypeptides comprisingthe amino acid sequences of the light chains and heavy chains of theantibodies of the invention. In one embodiment, the isolated polypeptidecomprises the sequence of any one of SEQ ID NOs: 1-33.

The term “polypeptide” is used in its conventional meaning, as asequence of amino acids. The polypeptides are not limited to a specificlength of the product. Peptides, oligopeptides, and proteins areincluded within the definition of polypeptide, and such terms can beused interchangeably herein unless specifically indicated otherwise.This term also includes post-expression modifications of thepolypeptide, for example, glycosylations, acetylations, phosphorylationsand the like, as well as other modifications known in the art, bothnaturally occurring and non-naturally occurring. A polypeptide can be anentire protein, or a subsequence thereof.

A polypeptide “variant,” as the term is used herein, is a polypeptidethat typically differs from a polypeptide specifically disclosed hereinin one or more substitutions, deletions, additions and/or insertions.Such variants can be naturally occurring or can be syntheticallygenerated, for example, by modifying one or more of the abovepolypeptide sequences of the invention and evaluating one or morebiological activities of the polypeptide as described herein and/orusing any of a number of techniques well known in the art.

For example, certain amino acids can be substituted for other aminoacids in a protein structure without appreciable loss of its ability tobind other polypeptides (for example, antigens) or cells. Since it isthe binding capacity and nature of a protein that defines that protein'sbiological functional activity, certain amino acid sequencesubstitutions can be made in a protein sequence, and, accordingly, itsunderlying DNA coding sequence, whereby a protein with like propertiesis obtained. It is thus contemplated that various changes can be made inthe peptide sequences of the disclosed compositions, or correspondingDNA sequences that encode said peptides without appreciable loss oftheir biological utility or activity.

In many instances, a polypeptide variant will contain one or moreconservative substitutions. A “conservative substitution” is one inwhich an amino acid is substituted for another amino acid that hassimilar properties, such that one skilled in the art of peptidechemistry would expect the secondary structure and hydropathic nature ofthe polypeptide to be substantially unchanged.

Amino acid substitutions generally are based on the relative similarityof the amino acid side-chain substituents, for example, theirhydrophobicity, hydrophilicity, charge, size, and the like. Exemplarysubstitutions that take various of the foregoing characteristics intoconsideration are well known to those of skill in the art and include:arginine and lysine; glutamate and aspartate; serine and threonine;glutamine and asparagine; and valine, leucine and isoleucine.

In another embodiment, the present invention provides an isolatedantigen comprising an epitope-scaffold that mimics the HIV-1 envelopespike epitope of broadly neutralizing antibody 8ANC195. On oneembodiment, the epitope-scaffold comprises a discontinous epitope and ascaffold, wherein the epitope is derived from HIV-1 gp120 and gp41, andwherein at least part of the scaffold is not derived from gp120 or gp41.In one embodiment, the discontinuous epitope comprises amino acidscorresponding to amino acid numbers 44-47. 90-94, 97, 234, 236-238, 240,274-278, 352-354, 357, 456, 463, 466. 487, and 625-641 of gp140 from HIVstrain 93TH057 numbered using standard numbering for HIV strain HXBC2 asdepicted in FIG. 20 and disclosed by Korber et al. (1998, Numberingpositions in HIV relative to HXBc2, p. 111-102-IV-103. In B. Korber, C.L. Kuiken, B. Foley, B. Hahn, F. McCutchan, J. W. Mellors, and J.Sodroski (ed.), Human retroviruses and AIDS. Los Alamos NationalLaboratories, Los Alamos, N. Mex.). In another embodiment, the aminoacids corresponding to amino acid numbers 234 and 276 are glycosylated.

Methods of making epitope-scaffolds are known in the art and disclosed,for example, by Correia. et al. Journal of Molecular Biology 405, 284(2011), Correia et. al. Structure 18, 1.1.16 (2010), Ofek et al, ProcNatl Acas Sci USA 107, 17780 (2010), MeLellan et al. J Mol Biol. 409,853 (2011), Azoitci et al. Science 334, 373 (2011) and inUS201010068217. Briefly, information obtained from the crystallographicanalysis disclosed herein is used to design epitope-scaffolds that mimicthe 8ANC195 epitope on the HIV-1 envelope spike. First, computationalmethods are utilized to identify non-HIV scaffold proteins capable ofsupporting the discontinuous epitope identified herein.Epitope-scaffolds are then designed and produced, and theirimmunological properties are characterized. For example, in the methodof Azoitei et al., the Protein Data Bank (www.pdb.org) is searched forsuitable scaffolds for the discontinuous epitope, for example by usingan algorithm such as Multigraft Match. An algorithm such as MultigraftDesign disclosed by Azoitei et al. is used for scaffold design in whichregions of the scaffold are deleted and new segments are built toconnect the epitope to the scaffold. Candidate epitope-scaffolds may beexpressed in a host cell and purified, and tested for binding to 8ANC195or another antibody that binds to the epitope recognized by 8ANC195.

The invention also includes isolated nucleic acid sequences encodingpart or all of the light and heavy chains of the described inventiveantibodies, and fragments thereof. Due to redundancy of the geneticcode, variants of these sequences will exist that encode the same aminoacid sequences. In one embodiment, the present invention provides anisolated nucleic acid encoding a polypeptide having the sequence of anyone of SEQ ID Nos:1-33. In another embodiment, the isolated nucleic acidencodes an antibody comprising a heavy chain comprising the sequence ofany one of SEQ ID Nos: 1-18 and a light chain comprising the sequence ofany one of SEQ ID Nos: 19-33.

The invention also includes isolated nucleic acid sequences that encodethe antigen comprising the epitope-scaffold of the invention.

The terms “nucleic acid” and “polynucleotide” are used interchangeablyherein to refer to single-stranded or double-stranded RNA, DNA, or mixedpolymers. Polynucleotides can include genomic sequences, extra-genomicand plasmid sequences, and smaller engineered gene segments thatexpress, or can be adapted to express polypeptides.

An “isolated nucleic acid” is a nucleic acid that is substantiallyseparated from other genome DNA sequences as well as proteins orcomplexes such as ribosomes and polymerases, which naturally accompany anative sequence. The term encompasses a nucleic acid sequence that hasbeen removed from its naturally occurring environment, and includesrecombinant or cloned DNA isolates and chemically synthesized analoguesor analogues biologically synthesized by heterologous systems. Asubstantially pure nucleic acid includes isolated forms of the nucleicacid. Accordingly, this refers to the nucleic acid as originallyisolated and does not exclude genes or sequences later added to theisolated nucleic acid by the hand of man.

A polynucleotide “variant,” as the term is used herein, is apolynucleotide that typically differs from a polynucleotide specificallydisclosed herein in one or more substitutions, deletions, additionsand/or insertions. Such variants can be naturally occurring or can besynthetically generated, for example, by modifying one or more of thepolynucleotide sequences of the invention and evaluating one or morebiological activities of the encoded polypeptide as described hereinand/or using any of a number of techniques well known in the art.

Modifications can be made in the structure of the polynucleotides of thedescribed invention and still obtain a functional molecule that encodesa variant or derivative polypeptide with desirable characteristics. Whenit is desired to alter the amino acid sequence of a polypeptide tocreate an equivalent, or even an improved, variant or portion of apolypeptide of the invention, one skilled in the art typically willchange one or more of the codons of the encoding DNA sequence.

Typically, polynucleotide variants contain one or more substitutions,additions, deletions and/or insertions, such that the immunogenicbinding properties of the polypeptide encoded by the variantpolynucleotide is not substantially diminished relative to a polypeptideencoded by a polynucleotide sequence specifically set forth herein.

In some embodiments, the polypeptide encoded by the polynucleotidevariant or fragment has the same binding specificity (i.e., specificallyor preferentially binds to the same epitope or HIV strain) as thepolypeptide encoded by the native polynucleotide. In some embodiments,the described polynucleotides, polynucleotide variants, fragments andhybridizing sequences, encode polypeptides that have a level of bindingactivity of at least about 50%, at least about 70%, and at least about90% of that for a polypeptide sequence specifically set forth herein.

The polynucleotides of the described invention, or fragments thereof,regardless of the length of the coding sequence itself, can be combinedwith other DNA sequences, such as promoters, polyadenylation signals,additional restriction enzyme sites, multiple cloning sites, othercoding segments, and the like, such that their overall length can varyconsiderably. A nucleic acid fragment of almost any length is employed.For example, illustrative polynucleotide segments with total lengths ofabout 10000, about 5000, about 3000, about 2000, about 1000, about 500,about 200, about i00, about 50 base pairs in length, and the like,(including all intermediate lengths) are included in manyimplementations of this invention.

Further included within the scope of the invention are vectors such asexpression vectors, comprising a nucleic acid sequence according to theinvention. Cells transformed with such vectors also are included withinthe scope of the invention.

The present invention also provides vectors and host cells comprising anucleic acid of the invention, as well as recombinant techniques for theproduction of a polypeptide of the invention. Vectors of the inventioninclude those capable of replication in any type of cell or organism,including, for example, plasmids, phage, cosmids, and mini chromosomes.In some embodiments, vectors comprising a polynucleotide of thedescribed invention are vectors suitable for propagation or replicationof the polynucleotide, or vectors suitable for expressing a polypeptideof the described invention. Such vectors are known in the art andcommercially available.

“Vector” includes shuttle and expression vectors. Typically, the plasmidconstruct also will include an origin of replication (for example, theColE1 origin of replication) and a selectable marker (for example,ampicillin or tetracycline resistance), for replication and selection,respectively, of the plasmids in bacteria. An “expression vector” refersto a vector that contains the necessary control sequences or regulatoryelements for expression of the antibodies including antibody fragment ofthe invention, in bacterial or eukaryotic cells.

As used herein, the term “cell” can be any cell, including, but notlimited to, that of a eukaryotic, multicellular species (for example, asopposed to a unicellular yeast cell), such as, but not limited to, amammalian cell or a human cell. A cell can be present as a singleentity, or can he part of a larger collection of cells. Such a “largercollection of cells” can comprise, for example, a cell culture (eithermixed or pure), a tissue (for example, endothelial, epithelial, mucosaor other tissue), an organ (for example, lung, liver, muscle and otherorgans), an organ system (for example, circulatory system, respiratorysystem, gastrointestinal system, urinary system, nervous system,integumentary system or other organ system), or an organism (e.g., abird, mammal, or the like).

Polynucleotides of the invention may synthesized, whole or in parts thatthen are combined, and inserted into a vector using routine molecularand cell biology techniques, including, for example, subcloning thepolynucleotide into a linearized vector using appropriate restrictionsites and restriction enzymes. Polynucleotides of the describedinvention are amplified by polymerase chain reaction usingoligonueleotide primers complementary to each strand of thepolynucleotide. These primers also include restriction enzyme cleavagesites to facilitate subcloning into a vector. The replicable vectorcomponents generally include, but are not limited to, one or more of thefollowing: a signal sequence, an origin of replication, and one or moremarker or selectable genes.

In order to express a polypeptide of the invention, the nucleotidesequences encoding the polypeptide, or functional equivalents, may beinserted into an appropriate expression vector, i.e., a vector thatcontains the necessary elements for the transcription and translation ofthe inserted coding sequence. Methods well known to those skilled in theart may be used to construct expression vectors containing sequencesencoding a polypeptide of interest and appropriate transcriptional andtranslational control elements. These methods include in vitrorecombinant DNA techniques, synthetic techniques, and in vivo geneticrecombination. Such techniques are described, for example, in Sambrook,J., et al. (1989) Molecular Cloning, A Laboratory Manual, Cold SpringHarbor Press, Plainview, N.Y., and Ausubel, F. M. et al. (1989) CurrentProtocols in Molecular Biology, John Wiley & Sons, New York. N.Y.

According to another embodiment, the present invention provides methodsfor the preparation and administration of an HIV antibody compositionthat is suitable for administration to a human or non-human primatepatient having HIV infection, or at risk of HIV infection, in an amountand according to a schedule sufficient to induce a protective immuneresponse against HIV, or reduction of the HIV virus, in a human.

According to another embodiment, the present invention provides methodsfor the preparation and administration of an HIV antigen compositionthat is suitable for administration to a human or non-human primatepatient having HIV infection, or at risk of HIV infection, in an amountand according to a schedule sufficient to induce a protective immuneresponse against HIV, or reduction of the HIV virus, in a human.

According to another embodiment, the present invention provides acomposition comprising at least one antibody or polypeptide of theinvention and a pharmaceutically acceptable carrier. The composition mayinclude a plurality of the antibodies having the characteristicsdescribed herein in any combination and can further include antibodiesneutralizing to HIV as arc known in the art. According to anotherembodiment, the present invention provides a composition comprising atleast one antigen of the invention and a pharmaceutically acceptablecarrier, can further include antibodies neutralizing to HIV as are knownin the art, and can further include an adjuvant.

It is to be understood that compositions can be a single or acombination of antibodies disclosed herein, which can be the same ordifferent, in order to prophylactically or therapeutically treat theprogression of various subtypes of HIV infection after vaccination. Suchcombinations can be selected according to the desired immunity. When anantibody or antigen is administered to an animal or a human, it can becombined with one or more pharmaceutically acceptable carriers,excipients or adjuvants as are known to one of ordinary skilled in theart. The composition can further include broadly neutralizing antibodiesknown in the art, including but not limited to, VRC01, b12, anti-V3loop, CD4bs, and CD4i antibodies as well as PG9/PG16-like antibodies.

Further, with respect to determining the effective level in a patientfor treatment of HIV, in particular, suitable animal models areavailable and have been widely implemented for evaluating the in vivoefficacy against HIV of various gene therapy protocols (Sarver et at(1993b), supra). These models include mice, monkeys and cats. Eventhough these animals are not naturally susceptible to HIV disease,chimeric mice models (for example, SCID, bg/nu/xid, NOD/SCID, SCID-hu,immunocompetent SCID-hu, bone marrow-ablated BALB/c) reconstituted withhuman peripheral blood mononuclear cells (PBMCs), lymph nodes, fetalliver/thymus or other tissues can be infected with lentiviral vector orHIV, and employed as models for HIV pathogenesis. Similarly, the simianimmune deficiency virus (SIV)/monkey model can be employed, as can thefeline immune deficiency virus (FIV)/cat model. The pharmaceuticalcomposition can contain other phannaceuticals, in conjunction with avector according to the invention, when used to therapeutically treatAIDS. These other pharmaceuticals can be used in their traditionalfashion (i.e., as agents to treat HIV infection).

According to another embodiment, the present invention provides anantibody-based pharmaceutical composition comprising an effective amountof an isolated antibody of the invention, or an affinity maturedversion, which provides a prophylactic or therapeutic treatment choiceto reduce infection of the HIV virus. According to another embodiment,the present invention provides an antigen-based pharmaceuticalcomposition comprising an effective amount of an isolated antigen of theinvention, which provides a prophylactic or therapeutic treatment choiceto reduce infection of the HIV virus. The pharmaceutical compositions ofthe present invention may be formulated by any number of strategiesknown in the art (e.g., see McGoff and Scher. 2000. Solution Formulationof Proteins/Peptides: in McNally, E. J. ed. Protein Formulation andDelivery. New York, N.Y.: Marcel Dekker; pp, 139-158; Akers andDefilippis, 2000, Peptides and Proteins as Parenteral Solutions. In:Pharmaceutical Formulation Development of Peptides and Proteins.Philadelphia, Pa.: Talyor and Francis; pp. 145-177; Akers, et al., 2002,Pharm. Biotechnol, 14:47-127). A pharmaceutically acceptable compositionsuitable for patient administration will contain an effective amount ofthe antibody in a formulation which both retains biological activitywhile also promoting maximal stability during storage within anacceptable temperature range. The pharmaceutical compositions can alsoinclude, depending on the formulation desired, pharmaceuticallyacceptable diluents, pharmaceutically acceptable carriers and/orpharmaceutically acceptable excipients, or any such vehicle commonlyused to formulate pharmaceutical compositions for animal or humanadministration. The diluent is selected so as not to affect thebiological activity of the combination. Examples of such diluents aredistilled water, physiological phosphate-buffered saline, Ringer'ssolutions, dextrose solution, and Hank's solution. The amount of anexcipient that is useful in the pharmaceutical composition orformulation of this invention is an amount that serves to uniformlydistribute the antibody throughout the composition so that it can beuniformly dispersed when it is to be delivered to a subject in needthereof. It may serve to dilute the antibody or antigen to aconcentration which provides the desired beneficial palliative orcurative results while at the same time minimizing any adverse sideeffects that might occur from too high a concentration. It may also havea preservative effect. Thus, for an active ingredient having a highphysiological activity, more of the excipient will be employed. On theother hand, for any active ingredient(s) that exhibit a lowerphysiological activity, a lesser quantity of the excipient will beemployed. Compositions comprising an antigen of the invention mayfurther comprise one or more adjuvants.

The above described antibodies and antibody compositions, comprising atleast one or a combination of the antibodies described herein, can beadministered for the prophylactic and therapeutic treatment of HIV viralinfection.

The above described antigens and antigen compositions, comprising atleast one or a combination of the antigens described herein, can beadministered for the prophylactic and therapeutic treatment of HIV viralinfection.

The present invention also provides kits useful in performing diagnosticand prognostic assays using the antibodies, polypeptides and nucleicacids of the present invention. Kits of the present invention include asuitable container comprising an HIV antibody, an antigen, a polypeptideor a nucleic acid of the invention in either labeled or unlabeled form.In addition, when the antibody, antigen, polypeptide or nucleic acid issupplied in a labeled form suitable for an indirect binding assay, thekit further includes reagents for performing the appropriate indirectassay. For example, the kit may include one or more suitable containersincluding enzyme substrates or derivatizing agents, depending on thenature of the label. Control samples and/or instructions may also beincluded. The present invention also provides kits for detecting thepresence of the HIV antibodies or the nucleotide sequence of the HIVantibody of the present invention in a biological sample by PCR or massspectrometry.

“Label” as used herein refers to a detectable compound or compositionthat is conjugated directly or indirectly to the antibody so as togenerate a “labeled” antibody. A label can also be conjugated to apolypeptide and/or a nucleic acid sequence disclosed herein. The labelcan be detectable by itself (for example, radioisotope labels orfluorescent labels) or, in the case of an enzymatic label, can catalyzechemical alteration of a substrate compound or composition that isdetectable. Antibodies and polypeptides of the described invention alsocan be modified to include an epitope tag or label, for example, for usein purification or diagnostic applications. Suitable detection meansinclude the use of labels such as, but not limited to, radionucleotides,enzymes, coenzymes, fluorescers, chemiluminescers, chromogens, enzymesubstrates or co-factors, enzyme inhibitors, prosthetic group complexes,free radicals, particles, dyes, and the like.

Methods for reducing an increase in HIV virus titer, virus replication,virus proliferation or an amount of an HIV viral protein in a subjectare further provided. According to another aspect, a method includesadministering to the subject an amount of an HIV antibody of theinvention effective to reduce an increase in HIV titer, virusreplication or an amount of an HIV protein of one or more HIV strains orisolates in the subject. According to another aspect, a method includesadministering to the subject an amount of an HIV antigen of theinvention effective to reduce an increase in HIV titer, virusreplication or an amount of an HIV protein of one or more HIV strains orisolates in the subject.

According to another embodiment, the present invention provides a methodof reducing viral replication or spread of HIV infection to additionalhost cells or tissues comprising contacting a mammalian cell with theantibody, or a portion thereof, which binds to the 8ANC195 antigenicepitope on gp120. According to another embodiment, the present inventionprovides a method of reducing viral replication or spread of HIVinfection to additional host cells or tissues comprising contacting amammalian cell with the antigen that mimics the 8ANC195 antigenicepitope on gp120.

According to another embodiment, the present invention provides a methodfor treating a mammal infected with a virus infection, such as, forexample, HIV, comprising administering to said mammal a pharmaceuticalcomposition comprising the HIV antibodies disclosed herein. According toone embodiment, the method for treating a mammal infected with HIVcomprises administering to said mammal a pharmaceutical composition thatcomprises an antibody of the present invention, or a fragment thereof.The compositions of the invention can include more than one antibodyhaving the characteristics disclosed (for example, a plurality or poolof antibodies). It also can include other HIV neutralizing antibodies asare known in the art, for example, but not limited to, VRC01, PG9 andb12.

Passive immunization has proven to be an effective and safe strategy forthe prevention and treatment of viral diseases. (See, for example,Keller et al., Clin. Microbiol. Rev. 13:602-14 (2000); Casadevall, Nat.Biotechnol. 20:114 (2002); Shibata et al., Nat. Med. 5:204-10 (1999);and igarashi et al., Nat. Med. 5:211-16 (1999). Passive immunizationusing human monoclonal antibodies provides an immediate treatmentstrategy for emergency prophylaxis and treatment of HIV.

According to another embodiment, the present invention provides a methodof inducing an HIV antigen-specific immune response in a mammal infectedwith HIV or at risk of infection with HIV comprising administering tothe mammal a pharmaceutical composition comprising the antigen of theinvention.

According to another embodiment, the present invention provides a methodof inducing an HIV antigen-specific immune response in a mammal infectedwith HIV or at risk of infection with HIV comprising; administering tothe mammal a pharmaceutical composition comprising a nucleic acidencoding the antigen of the invention.

Subjects at risk for HIV-related diseases or disorders include patientswho have come into contact with an infected parson or who have beenexposed to HIV in some other way. Administration of a prophylactic agentcan occur prior to the manifestation of symptoms characteristic ofHIV-related disease or disorder, such that a disease or disorder isprevented or, alternatively, delayed in its progression.

For in vivo treatment of human and non-human patients, the patient isadministered or provided a pharmaceutical formulation including an HIVantibody or antigen of the invention. When used for in vivo therapy, theantibodies and antigens of the invention are administered to the patientin therapeutically effective amounts (i.e., amounts that eliminate orreduce the patient's viral burden). The antibodies or antigens areadministered to a human patient, in accord with known methods, such asintravenous administration, for example, as a bolus or by continuousinfusion over a period of time, by intramuscular, intraperitoneal,intracerobrospinal, subcutaneous, intra-articular, intrasynovial,intrathecal, oral, topical, or inhalation routes. The antibodies can beadministered parenterally, when possible, at the target cell site, orintravenously. In some embodiments, antibody is administered byintravenous or subcutaneous administration. Therapeutic compositions ofthe invention may be administered to a patient or subject systemically,parenterally, or locally. The above parameters for assessing successfultreatment and improvement in the disease are readily measurable byroutine procedures familiar to a physician.

For parenteral administration, the antibodies or antigens may beformulated in a unit dosage injectable form (solution, suspension,emulsion) in association with a pharmaceutically acceptable, parenteralvehicle. Examples of such vehicles include, but are not limited, water,saline, Ringer's solution, dextrose solution, and 5% human serumalbumin. Nonaqueous vehicles include, but are not limited to, fixed oilsand ethyl oleate. Liposomes can be used as carriers. The vehicle maycontain minor amounts of additives such as substances that enhanceisotonicity and chemical stability, such as, for example, buffers andpreservatives. The antibodies can be formulated in such vehicles atconcentrations of about 1 mg/ml to 10 mg/ml.

The dose and dosage regimen depends upon a variety of factors readilydetermined by a physician, such as the nature of the infection, forexample, it therapeutic index, the patient, and the patient's history.Generally, a therapeutically effective amount of an antibody or antigenis administered to a patient. In some embodiments, the amount ofantibody or antigen administered is in the range of about 0.1 mg/kg toabout 50 mg/kg of patient body weight. Depending on the type andseverity of the infection, about 0.1 mg/kg to about 50 mg/kg body weight(for example, about 0.1-15 mg/kg/dose) of antibody is an initialcandidate dosage for administration to the patient, whether, forexample, by one or more separate administrations, or by continuousinfusion. The progress of this therapy is readily monitored byconventional methods and assays and based on criteria known to thephysician or other persons of skill in the art. The above parameters forassessing successful treatment and improvement in the disease arereadily measurable by routine procedures familiar to a physician.

A dosage regimen for administration of an antigen to a patient may be asuitable immunization regimen, including for example at least threeseparate inoculations. The second inoculation may be administered morethan at least two weeks after the first inoculation. The thirdinoculation may be administered at least several months after the secondadministration.

Other therapeutic regimens may be combined with the administration ofthe HIV antibody or antigen of the present invention. The combinedadministration includes co-administration, using separate formulationsor a single pharmaceutical formulation, and consecutive administrationin either order, wherein preferably there is a time period while both(or all) active agents simultaneously exert their biological activities.Such combined therapy can result in a synergistic therapeutic effect.The above parameters for assessing successful treatment and improvementin the disease are readily measurable by routine procedures familiar toa physician.

The terms “treating” or “treatment” or “alleviation” are usedinterchangeably and refer to both therapeutic treatment and prophylacticor preventative measures; wherein the object is to prevent or slow down(lessen) the targeted pathologic condition or disorder. Those in need oftreatment include those already with the disorder as well as those proneto have the disorder or those in whom the disorder is to be prevented. Asubject or mammal is successfully “treated” for an infection if, afterreceiving a therapeutic amount of an antibody or antigen according tothe methods of the present invention, the patient shows observableand/or measurable reduction in or absence of one or more of thefollowing: reduction in the number of infected cells or absence of theinfected cells; reduction in the percent of total cells that areinfected; and/or relief to some extent, one or more of the symptomsassociated with the specific infection; reduced morbidity and mortality,and improvement in quality of fife issues. The above parameters forassessing successful treatment and improvement in the disease arereadily measurable by routine procedures familiar to a physician.

The term “therapeutically effective amount” refers to an amount of anantibody or a drug effective to treat a disease or disorder in a subjector mammal.

Administration “in combination with” one or more further therapeuticagents includes simultaneous (concurrent) and consecutive administrationin any order.

“Carriers” as used herein include pharmaceutically acceptable carriers,excipients, or stabilizers that are nontoxic to the cell or mammal beingexposed thereto at the dosages and concentrations employed. Often thephysiologically acceptable carder is an aqueous pH buffered solution.Examples of physiologically acceptable carriers include, but not limitedto, buffers such as phosphate, citrate, and other organic acids;antioxidants including, but not limited to, ascorbic acid; low molecularweight (less than about 10 residues) polypeptide; proteins, such as, butnot limited to, serum albumin, gelatin, or immunoglobulins; hydrophilicpolymers such as, but not limited to, polyvinylpyrrolidone; amino acidssuch as, but not limited to, glycine, glutamine, asparagine, arginine orlysine; monosaccharides, disaccharides, and other carbohydratesincluding, but not limited to, glucose, mannose, or dextrins; chelatingagents such as, but not limited to, EDTA; sugar alcohols such as, butnot limited to, mannitol or sorbitol; salt-forming counterions such as,but not limited to, sodium; and/or nonionic surfactants such as, but notlimited to, TWEEN; polyethylene glycol (PEG), and PLURONlC'S.

According to another embodiment, the present invention providesdiagnostic methods. Diagnostic methods generally involve contacting abiological sample obtained front a patient, such as, for example, blood,serum., saliva, urine, sputum, a cell swab sample, or a tissue biopsy,with an HIV antibody and determining whether the antibody preferentiallybinds to the sample as compared to a control sample or predeterminedcut-off value, thereby indicating the presence of the HIV virus.

According to another embodiment, the present invention provides methodsto detect the presence of the HIV antibodies of the present invention ina biological sample from a patient. Detection methods generally involveobtaining a biological sample from a patient, such as, for example,blood, serum, saliva, urine, sputum, a cell swab sample, or a tissuebiopsy and isolating HIV antibodies or fragments thereof, or the nucleicadds that encode an HIV antibody, and assaying for the presence of anHIV antibody in the biological sample. Also, the present inventionprovides methods to detect the nucleotide sequence of an HIV antibody ina cell. The nucleotide sequence of an HIV antibody may also be detectedusing the primers disclosed herein. The presence of the HIV antibody ina biological sample from a patient may be determined utilizing knownrecombinant techniques and/or the use of a MSS spectrometer.

According to another embodiment, the present invention provides methodsfor detecting or isolating an HIV-1 binding antibody in a subjectcomprising obtaining a biological sample from the subject, contactingsaid sample with the antigen of the invention, and conducting an assayto detect or isolate an HIV-1 binding antibody.

The term “assessing” includes any form of measurement, and includesdetermining if an element is present or not. The terms “determining”,“measuring”, “evaluating”, “assessing” and “assaying” are usedinterchangeably and include quantitative and qualitative determinations.Assessing may be relative or absolute. “Assessing the presence of”includes determining the amount of something present, and fordetermining whether it is present or absent. As used herein, the terms“determining,” “measuring,” and “assessing,” and “assaying” are usedinterchangeably and include both quantitative and qualitativedeterminations.

Where a value of ranges is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range is encompassed within the invention. The upper and lowerlimits of these smaller ranges which may independently be included inthe smaller ranges is also encompassed within the invention, subject toany specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either bothof those included limits are also included in the invention.

EXAMPLE 1

This example describes materials and methods used in EXAMPLES 2-5 below.

Protein expression and purification

The antibodies used in this study were produced and purified as inpreviously-described studies (Diskin et al., Science 334, 1289 (2011).)Briefly, 8ANC195, 3BNC60 and chimeric antibody (mature HC/various LC;δ/κ combinations of newly isolated 8ANC195 variants) IgGs were expressedby transiently transfecting HEK293-6E cells with vectors containing theappropriate heavy and light chain genes. Secreted IgGs were purifiedfrom cell supernatants using protein A affinity chromatography (GEHealthcare). For neutralization assays. IgGs were diluted to 1 mg/mLstocks in 20 mM Tris pH 8.0, 150 mM sodium chloride (TBS buffer).8ANC195 Fab was expressed with a 6×-His tag on the C-terminus of C_(H)1as described for IgGs and purified using Ni²⁻-NTA affinitychromatography (GE Healthcare) and Superdex 200 16/60 size exclusionchromatography (GE Healthcare).

A truncated gp120 from the HIV-1 strain 93TH057 containing mutationsAsn88GIn_(gp120), Asn289GIn_(gp120), Asn334GIn_(gp120),Asn392GIn_(gp120), Asn448GIn_(gp120) was produced by transientlytraasfecting HEK293-S (GnTI-/−) cells adapted for growth in suspensionby the Cattech Protein Expression Center with a pTT5 vector encodingHis-tagged gp120. Secreted gp120 was captured on Ni²⁻-NTA resin (GEHealthcare) and further purified using Superdex 200 16/60 size exclusionchromatography (GE Healthcare).

Soluble CD4 domains 1 and 2 (sCD4) and sCD4_(K75T) were produced asdescribed previously (Diskin et al. Nat Struct Mol Biol 17, 608 (2010)).Briefly, the pACgp67b vector encoding 6×-His-tagged sCD4 or sCD4_(K75T)(residues 1-186 of mature CD4) was used to make infectious baculovirusparticles using BacoloGold (BD Biosynthesis). Protein was expressed inHi5 cells, captured on a Ni²⁻-NTA column (GE Healthcare) and furtherpurified using Superdex 200 16/60 size exclusion chromatography (GEHealthcare). To remove an N-linked glycan introduced by mutation insCD4_(K75T), the protein was treated with Endoglycosidase H (New EnglandBiolabs) for 16 hours at 25° C. and then purified by Superdex 200 16/60size exclusion chromatography (GE Healthcare).

For complex crystallization trials, purified 8ANC195 Fab, 93TH057 gp120and EndoH-treated sCD4_(K75T) were incubated at a 1:1:1 molar ratio for2 hours at 25 C. The complex was purified by Superdex 200 10/300 sizeexclusion chromatography (GE Healthcare) and the peak corresponding to8ANC195 Fab/gp120/sCD_(K75T) complex concentrated to 16 mg/mL in TBSbuffer. For crystallization of 8 ANC195 Fab alone, the protein wasconcentrated to 20 mg/mL in TBS buffer.

Purified BG505 SOSIP trimers (Julien et al., PLoS pathogens 9, e1003342(2013); Lyumkis et al., Science 342, 1484 (2013); Sanders et al., PLoSpathogens 9, e1003618 (2013)) for EM studies were the gift of Dr. JohnP. Moore (Weill Cornell Medical College).

Crystallization

Crystallization conditions were screened using vapor diffusion insitting drops set using a Mosquito® crystallization robot (TTP labs) ina final volume of 200 nL per drop (1:1 protein to reservoir ratio)utilizing commercially available crystallization screens (HamptonResearch, Microlytic). Initial crystallization hits for 8ANC195 Fab andfor 8ANC195 Fab/93TH057 gp120/sCD4_(K75) complex were identified usingthe MCSG-1 (Microlytic) and PEGRx (Hampton) screens and then manuallyoptimized. Crystals of 8ANC195 Fab (space group P4₁2₁2, a=66.5 Å, b=66.5Å, c=219.0 Å; one molecule per asymmetric unit) were obtained uponmixing a protein solution at 11 mg/mL with 0.1M Hepes pH 7, 20% PEG6,000, 10 mM zinc chloride at 20° C. Crystals were briefly soaked inmother liquor solution supplemented with 20% ethylene glycol beforeflash cooling in liquid nitrogen. Crystals of the 8ANC195 Fab/93TH057gp120/sCD4_(K75T) complex (space group P2₁2₁2₁, a=66.5 Å, b=132.5 Å,c=142.8 Å; one molecule per asymmetric unit) were obtained upon mixing aprotein solution at 16 mg: Int with 14% polyethylene glycol 3,350, 0.1 MHEPES pH 7.3, 2% benzamidine HCl at 20′C. Crystals were briefly soakedin mother liquor solution supplemented with 30% ethylene glycol beforeflash cooling in liquid nitrogen.

Crystallographic Data Collection, Structure Solution and Refinement

X-ray diffraction data for 8ANC195 Fab crystals were collected at theArgonne National Laboratory Advanced Photon Source (APS) bean 23-ID-Dusing a MAR 300 CCD detector. X-ray diffraction data for 8ANC195Fab/93TH057 gp120/sCD4_(K75T) complex crystals were collected at theStanford Synchrotron Radiation Lightsource (SSRL) beamline 12-2 using aPilatus 6M pixel detector (Dectris). The data were indexed, integratedand scaled using XDS (Kabsch, Acta Crystallogr D Biol Crystallogr 66,133 (2010)).

The 8ANC195 Fab structure was solved by molecular replacement usingPhenix (Adams et al., Acta Crystallogr D Biol Crystallogr 66. 213(2010)) and the V_(H)V₁ and C_(H)1C_(L) domains of NIH-45-46 Fab (PDBcode 3U7W) lacking all CDR loops as two separate search models. Themodel was then refined to 2.13 Å resolution using an iterative approachinvolving refinement and verification of model accuracy with simulatedannealing composite omit maps using the Phenix crystallography package,and manually fitting models into electron density maps using Coot(Emsley et al., Acta Crystallogr D Biol Crystallor 60, 2126 (2004). Thefinal model (R_(work)=21.4%; R_(free)=25.7%) includes 3,279 proteinatoms and 127 water molecules as shown in Table 1.

TABLE 1 BANC195 Fab/go120/ sCD4 complex BANC195 Fab Data collectionResolution range (Å) 39.22-3.0 (3.22-3.0)  29.73-2.1 (2.21-2.1)  Spacegroup P 2₁ 2₁ 2₁ P 4₁ 2₁ 2 Cell dimensions a, b, c (Å) 66.53, 132.49,142.77 66.48, 66.46, 219.03 α, β, γ (°) 90, 90, 90 90, 90, 90 Totalreflections 229212 (12539)  239217 (24708)  Unique reflections 36730(3064)  28097 (2786)  Multiplicity 6.2 (6.3) 8.4 (6.9) Completeness (%)97.65 (98.80) 98.92 (91.00) Mean I/o(I) 7.86 (2.1)  11.90 (3.16)  WilsonB-factor 61.95 32.47 R_(merge) 0.1747 (0.765)  0.1225 (0.5802) CC1/20.996 (0.864) 0.996 (0.876) CC* 0.999 (0.980) 0.999 (0.966) RefinementR_(work)/R_(free) 0.2655/0.3149 0.2431/0.2772 Number of atoms 7272 3311Protein 6881 3311 Ligands 391 0 Water 0 0 Protein residues 939 437 RMS(bonds) 0.023 0.008 RMS (angles) 1.33 1.17 Clashscore 22.78 12.43Average B-factor 81.5 36.5 Protein 81 36.5 Ligands 89 — Water — —Statistics for the highest-resolution shell are shown in parentheses.

96.7S%, 2.78% and 0.0% of the residues were in the favored, allowed anddisallowed regions, respectively, of the Ramachandran plot. Disorderedresidues that were not included in the model include residues 146-153,233-238 and the 6×-His tag of the 8ANC195 heavy chain, and residues214-215 of the lipht chain.

The 8ANC195 Fab/93TH057 gp120/sCD4_(K75T) complex structure was solvedby, molecular replacement using Phaser (Adams et al., Acta Crystallogr DBiol Crystallogr 66, 213 (2010)) and the V_(H)V_(L), and C_(H)1C_(L),domains of 8ANC195 (lacking all CDR loops), 93TH057 gp120 (taken fromPOB code 3U7Y), and sCD4 (taken from PDB code 3LQA) as separate searchmodels. The complex structure was refined to 3.0 Å resolution asdescribed for the Fab structure. In addition to considering I/σ₁ andcompleteness of the highest resolution shell (2.1% and 99.9%,respectively), CC_(1/2) statistic (Karplus et al., Science 336. 1030(2012)) (correlation coefficient between two random halves of the dataset where CC_(1/2)>10%) was used to determine the high-resolution cutofffor the data. Phenix was used to compute CC_(1/2) (85.4% for the highestresolution shell and 99.8% for the entire data set), supporting ourhigh-resolution cutoff determination.

The final model (R_(work)=23.4%; R_(free)=28.6%) includes x proteinatoms and y atoms of carbohydrates (Table S1). 96.2%, 3.8% and 0.0% ofthe residues were in the favored, allowed and disallowed regions,respectively, of the Ramachandran plot. Disordered residues that werenot included in the model include residues 146-153, 206-208, 233-238 andthe 6×-His tag of the 8ANC195 heavy chain, residues 213-215 of the lightchain, residues 125-197 (V1/V2 substitution), 302-324 (V3 substitution), residues 397-409 (a total of 6 residues from V4), residues 492-494and the 6×-His tag of 93TH057 gp120 and residues 106-111, 154-155,177-186 of sCD4_(K75T).

Buried surface areas were calculated using PDBePISA (Krissinel et al.,Journal of molecular biology 372, 774 (2007)) and a 1.4 Å probe.Superimposition calculations were done and molecular representationswere generated using PyMol (Schrödinger (The PyMOL Molecular GraphicsSystem, 2011). Pairwise Cα alignments were performed using PDBeFold(Krissinel al., Acta Crystallogr D Biol Crystaliogr 60, 2256 (2004)).

ELISAs

High-binding 96-well ELISA plates (Costar) were coated overnight with 5μg/well of purified gp120 in 100 mM sodium carbonate pH 9.6. Afterwashing with TBS containing 0.05% Tween 20, the plates were blocked for2 h with 1% BSA, 0.05% Tween-TBS (blocking buffer) and then incubatedfor 2 h with 8ANC195 lgG (1 μg/mL) mixed with 1:2 serially dilutedsolutions of potential antibody competitors (sCD4, J3 VHH, 3BNC60 Fab,NIH45-46 Fab) in blocking buffer (competitor concentration range from 5to 320 μg/mL). After washing with TBS containing 0.05% Tween 20, theplates were incubated with HRP-conjugated goat anti-human IgG antibodies(Jackson ImmunoReseach) (at 0.8 μg/ml in blocking buffer) for 1 hour.The ELISAs were developed by addition of HRP chromogenic substrate (TMBsolution, BioLegend) and the color development stopped by addition of10% sulfuric acid. Experiments were performed in duplicate.

Surface Plasmon Resonance

Experiments were performed using a Biacore T100 (Biacore) using astandard single-cycle kinetics method. YU-2 and 93TH057 gp120 proteinswere primary amine-coupled on CMN5 chips (Biacore) at a coupling densityof 1,000 RUs and one flow cell was mock coupled using HBS-EP+ buffer.8ANC195 and chimeric IgGs were injected over flow cells at increasingconcentrations (62.5 to 1,000 nM), at flow rates of 20 μl/min with 5consecutive cycles of 2 min association/1 min dissociation and a final10 min dissociation phase. Flow cells were regenerated with 3 pulses of10 mM glycine pH 2.5. Apparent binding constants (K_(D) (M)) werecalculated from single-cycle kinetic analyses after subtraction ofbackgrounds using a 1:1 binding model without a bulk reflective index(RI) correction (Biacore T100 Evaluation software). Binding constantsfor bivalent IgGs are referred to as “apparent” affinities to emphasizethat the K_(D) values include potential avidity effects.

Neutralization Assays

A TZM-bl/pseudovirus neutralization assay was used to evaluate theneutralization potencies of the antibodies as described (Montefiori,Current protocols in immunology edited by John. E. Coligan etal.,Chapter 12, Unit 12 11 (2005)). Pseudoviruses were generated by cotransfection of HEK 293T cells with an Env expression plasmid and areplication-defective backbone plasmid. Neutralization was determined bymeasuring the reduction in luciferase reporter gene expression in thepresence of antibody following a single round of pseudovims infection inTZM-bi cells. Nonlinear regression analysis was used to calculate theconcentrations at which half-maximal inhibition was observe (IC₅₀values).

Negative-stain EM

The BG505 SOSIP.664/8ANC195 Fab complex and grids were prepared asdescribed previously (Kong et al., Nat Struct Mol Biol 20, 796 (2013).The data were collected on an FE1 Tecnai T12 electron microscope coupledwith a Tietz TemCam-F416 4k×4k CMOS camera using the LEGINON interface.Images were collected in 10° increments from 0° to −40° using, a defocusrange of 0.6-0.9 μm at a magnification of 52,000×, resulting in a pixelsize of 2.05 Å at the specimen plane. Particles were selected usingDogPicker (Voss et al., Journal of structural biology 166, 205 (2009))within the Appion software package (Lander et al., Journal of structuralbiology 166, 95 (2009)), and sorted from reference-free 2D classaverages using the SPARX package (Penczek et al, Ultramicroscopy 40, 33(1992). An initial model was generated by common lines from classaverages using the EMAN2 package (Tang et al., Journal of structuralbiology 157, 38 (2007) and was refined using 11,637 unbinned particles.The refinement was carried out using the SPARX package (Penezek et al.,Ultramiscroscopy 53, 251 (1994)) with C3 symmetry applied. The resultingresolution at a 0.5 Fourier Shell Correlation (FSC) cut-off was 18.7 Å(FIGS. 6A,B).

Human Samples

Human samples were collected after signed informed consent in accordancewith Institutional Review Board (IRB)-reviewed protocols by allparticipating institutions. Patient 8 was selected from a cohort ofelite controllers that were followed at the Raton Institute in Boston.

Isolation of 8ANC195 Variants

Single Cell clonal variants of 8ANC195 were isolated by 2CC.core-specific single cell sorting, followed by reverse transcription andimmunoglobulin gene amplification as described previously (Scheid etal., Science 333, 1633 (2011)). Immunoglobulin genes were cloned intoheavy and light chain expression vectors and co-transfected for IgGproduction as described previously (Tiller et al.., Journal ofImmunological methods 329, 112 (2008)).

IgG+ CD19+ memory B cells were bulk sorted on a FACS AriaIII cellsorter. Bulk mRNA was extracted using TRIzol (invitrogen) and reversetranscribed as previously described (Scheid et al., Science 333. 1633t2011)). 8ANC195-related heavy and light chain genes were PCR amplifiedusing the following clone-specific primers:

For heavy chain amplification: (SEQ ID NO: 50)  5′GGTGTACATTCTCAGATACACCTCGTACAA 3′ and (SEQ ID NO: 51) 5′CAGGTGTCCAGTCTCAGATACA 3′ as forward primers and (SEQ ID NO: 57) 5′GCGGAGACGGAGATGAGGGTT 3′ as a reverse primer.For light chain amplification: (SEQ ID NO: 52) 5′GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTATA GGT 3′ and (SEQ ID NO: 53)5′ GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCGCATCT 3′ as forward and(SEQ ID NO: 58) 5′ GTTTCACCTCAACTTTAGTCCCTT 3′ as well as(SEQ ID NO: 59) 5′ GTTTCACCTCAACTTTAGTCCCTTGGCCGAAGGTC 3′ asreverse primers.

Amplification products were gel purified and cloned into TOPO TAsequencing vectors (Invitrogen) and expression vectors as describedpreviously (T. Tiller et al., Journal of immunoloolcal methods 329, 112(2008)),

Phylogenetie Tree and Alignment Assembly.

Phylogenetie trees were assembled using Geneious Neighbor-Joining TreeSoftware. Sequence Alignments were performed using DNA Star (Instal Walignment software.

Computational Analysis.

The program Antibody Database (West, Jr, et at, Proceedings of theNational Academy of Sciences of the United States of America 1.10, 10598(2013)) was used to analyze 8ANC195 neutralization panel data fromScheid et al., Science 333, 1633 (2011) and Chuang et al. Journal ofvirology 87, 10047 (2013). This method attempts to model the variationin neutralization potency across strains based on a sum of terms(“rules”) corresponding to specific residues or potential N-linkedglycosylation site (PNGS) positions. With the free residual optiondeselected, the analysis finds a rule corresponding to ˜3-fold better8ANC195 neutralization for strains with Glu632_(gp41). This correlationappears to hold across Glades based on neutralization data for strainshaving the most favorable glycosylation pattern (PNGS at 234_(gp120) and276_(gp120), and not at 230_(gp120) (22). For all clades, the residue at632_(gp41) versus geometric mean IC₅₀s for 8ANC195 on strains with themost favorable glycosylation pattern was as follows: Glu, 0.43 μg/mL(n=53) versus Asp, 1.31 μg/mL (n=51). For separate clades, thecorrelations were Clade A: Glu, 0.47 μg/mL (n=3); Asp, 1.30 μg/mL(n=24); Clade B: Glu, 0.18 μg/mL (n=15); Asp, 0.72 μg/mL (n=6); Clade C:Glu, 0.32 μg/mL (n=2); Asp, 1.31 μg/mL (n=20).

Statistical Analysis of Neutralization Potencies of 8ANC195Variants

IC₅₀ values derived from neutralization assays with 8ANC195 and itsδ52_(HC)/κ5_(LC) variant against 11 sensitive virus strains (IC₅₀≤50)were analyzed by G-test for the relationship between the amino acididentity at position 636_(gp41) and the antibody IC₅₀. For each antibodythe partition between “high” and “low” IC₅₀s was chosen such thatapproximately half of the strains had high IC₅₀s (0.8 μ/mL for 8ANC195,0.1 μg/mL for δ52_(HC)/κ5_(LC)).

EXAMPLE 2

Determination of the Crystal Structure of the Fab Fragment of 8ANC195alone and Complexed with HIV-1 gp120 Core and CD4 Domains 1-2 (sCD4)

To determine the epitope recognized by 8ANC195 and investigate itsneutralization mechanism, crystal structures were solved of the Fabfragment of 8ANC195 alone and complexed with an HIV-1 clade A/E 93TH057gp120 core and CD4 domains 1-2 (sCD4) at 1.9 Å and 2.9 Å resolution,respectively (FIGS. 16A,B; Table 1). Five PNGSs on the core gp120 wereremoved by mutation (Asn88GIn_(gp120), Asn289GIn_(gp120),Asn334GIn_(gp120), Asn392GIn₁₂₀, Asn448GIn_(gp120)) to reduce glycanheterogeneity.

Comparison of 8ANC195 Fab in its free versus gp120-bound states revealedhigh structural similarity (RMSD=0.7 Å for 236 Cα atoms of V_(H)-V_(L))except for a 3.5 Å displacement of the loop connecting strands D and Ein HC FWR3 (FIG. 16A). The CDRH1 and CDRH3 loops were folded intohook-like tertiary structures in free and gp120-bound Fabs; thereforethe conformations were not induced upon binding to gp120 (FIG. 16A andFIGS. 2A,B). The CDRH3 architecture differed from CDRH3s in otherantibodies including anti-HIV-1 antibodies with long CDR loops (FIG.2C). The CDRH1 loop conformation was stabilized by a hydrogen bondnetwork among backbone atoms of CDRH1, burial of Phe30_(HC), andhydrogen bonds with Asp73_(HC) and Thr104_(HC) (FIG. 2A). CDRH3 had acomplex tertiary structure in which residues 102_(HC)-110_(HC) formed aloop protruding ˜10 Å from the antibody surface, and residues111_(HC)-118_(HC) formed a β-sheet subdomain that was stabilized byhydrophobic stacking between His113_(HC) and Trp33_(LC) and a hydrogenbond between Met117_(HC) and GIn90_(LC) (FIG. 2B). The side chain ofTyr92_(LC) hydrogen bonded with the Gly110_(HC) carbonyl oxygen,stabilizing a kink in the loop that formed the transition between thesesecondary structure elements (FIG. 2B),

The complex structure showed independent binding of sCD4 and 8ANC195 Fabto distinct sites on gp120 (FIG. 16B). sCD4 interacted with the gp120core as in other sCD4-gp120 structures (K wong et al., Nature 393, 648(1998)) (FIG. 3A), thus its binding was not altered by the presence ofthe adjacent antibody, consistent with binding and neutralizationexperiments showing no effects of CD4 addition on 8ANC195 activity (FIG.3B,C). sCD4 did, however, contribute to crystal packing (FIG. 3D),rationalizing why diffraction-quality crystals failed to grow in itsabsence. In the ternary complex structure, 8ANC195 bound to a gp120region adjacent to the CD4 binding site, contacting mainly the gp120inner domain, loops D and V5, and a small patch of the gp120 outerdomain (His352_(gp120)-Asn354_(gp120)) (FIG. 16 B,C).

8ANC195 Fab bound gp120 core exclusively with its HC, using residues inFWRs and its three CDR loops to form an extensive interface (3,671 Å²total buried surface area; 1287 Å² HC-gp120 protein contacts; 2,384 Å²HC-gp120 glycan contacts) (FIG. 16B, 2, FIG. 4; Table 2).

Buried Surface Area (BSA) at Interfaces Hydrogen Bonds at Interfacesgp120 BSA (A²) 8ANC195 HC BSA (A²) gp120 8ANC195 HC Distance (A) VAL 4419.4 ASN 28 28.6 THR 278 Oγ1 THR 75 O 2.43 TRP 45 17.4 THR 29 22.9 ARG456 NH2 GLY 76 O 3.37 LYS 46 35.1 GLY 31 26.3 ASN 354 Nδ2 SER 77 Oγ 2.88ASP 47 39.7 LEU 32 56.0 THR 278 Oγ1 SER 78 O 3.34 THR 90 39.1 ARG 5454.5 ASN 92 Nδ2 THR 104 O 2.88 GLU 91 5.7 TRP 55 4.2 ASN 92 Nδ2 TYR 105O 3.08 ASN 92 87.3 LYS 56 4.4 HIS 352 O THR 75 Oγ1 3.14 PHE 93 9.0 LEU74 65.7 ASN 354 Oδ1 THR 75 Oγ1 3.08 ASN 94 38.0 THR 75 44.3 ASP 47 Oδ2TYR 105 OH 3.09 LYS 97 7.2 GLY 76 81.0 LYS 487 Nζ TYR 105 OH 3.49 THR236 27.7 SER 77 33.9 GLY 237 21.1 SER 78 5.5 PRO 238 51.6 PRO 79 3.9 LYS240 4.1 THR 104 11.7 SER 274 0.2 TYR 105 100.2 GLU 275 7.0 ASP 106 16.8ASN 276 15.9 LYS 107 24.0 LEU 277 37.8 TRP 108 70.3 THR 278 69.6 HIS 35217.0 PHE 353 10.0 ASH 354 48.1 LYS 357 3.5 ARG 456 13.8 THR 463 0.2 GLU466 0.1 LYS 487 8.0 Total gp120 633.4 Total 8ANC195 HC 653.9 gp120 BSA(A²) 8ANC195 HC BSA (A²) gly276 NAG¹ 121.2 TYR 25 12.8 GLY 26 36.7 VAL27 6.5 ASM 28 15.3 LEU 74 21.2 PRO 79 4.2 gly276 NAG² 100.5 GLN 1 18.8HIS 3 8.0 TYR 25 40.8 GLY 26 12.8 gly276 BMA³ 65.5 HIS 3 35.3 VAL 5 6.5TYR 25 19.4 gly276 MAN⁴ 68.5 GLN 1 14.9 ILE 2 1.6 HIS 3 40.0 gly276 MAN⁵45.2 VAL 5 18.4 TYR 25 18.7 Total gly276 398.8 Total 8ANC195 HC 331.8Buried Surface Area (BSA) at Interfaces Hydrogen Bonds at Interfacesgp120 BSA (A²) 8ANC195 HC BSA (A²) gp120 8ANC195 HC Distance (A) gly234NAG¹ 108.4 ASN 28 1.5 gly234 NAG¹ O4 TRP 55 Nδ1 2.99 THR 29 9.8 gly234NAG¹ O3 ASP 73 Oδ2 3.30 TRP 55 24.9 gly234 NAG² O6 ASP 73 N 3.13 ASP 7329.7 gly234 MAN⁵ O6 VAL 72 N 2.75 LEU 74 16.8 gly234 MAN⁵ O6 ILE 81 O2.64 gly234 NAG² 128.4 ARG 54 1.0 gly234 MAN⁸ O3 GLU 85 Oγ2 3.17 TRP 5553.4 gly234 MAN⁸ O4 GLU 85 Oγ2 2.34 ALA 71 2.8 gly234 MAN¹⁰ O3 ALA 59 N3.50 VAL 72 11.6 gly234 MAN¹⁰ O2 ALA 59 N 3.03 ASP 73 13.1 gly234 MAN¹⁰O6 VAL 67 O 3.17 gly234 BMA³ 100.6 ILE 52 9.5 gly234 MAN¹⁰ O6 GLY 65 N2.58 TRP 55 24.5 gly234 MAN¹⁰ O2 SER 68 O 3.28 LYS 56 0.2 gly234 MAN¹⁰O2 SER 58 N 3.19 SER 57 3.4 gly234 MAN¹⁰ O3 SER 57 O 2.79 ILE 69 0.8 SER70 10.5 ALA 71 11.4 VAL 72 10.5 gly234 MAN⁴ 55.2 SER 70 8.8 ALA 71 5.2VAL 72 31.0 gly234 MAN⁵ 129.8 SER 70 11.9 ALA 71 1.6 VAL 72 18.1 ILE 8118.5 SER 83 18.6 gty234 MAN⁶ 118.4 THR 19 16.7 LEU 68 24.3 SER 70 8.9SER 83 7.6 GLU 85 25.7 gly234 MAN⁷ 84.2 ILE 52 3.3 TRP 55 13.4 LYS 561.8 SER 57 20.2 LEU 68 16.2 ILE 69 3.8 SER 70 2.1 gly234 MAN⁸ 68.3 SER57 30.2 SER 58 0.2 VAL 67 2.6 LEU 68 24.4 ILE 69 1.2 gly234 MAN¹⁰ 198.7SER 57 20.9 SER 58 11.0 ALA 59 21.8 ARG 64 14.3 GLY 65 14.2 VAL 67 13.3LEU 68 10.9 ILE 69 3.5 Total gly234 992.0 Total 8ANC195 HC 661.2

A loop in FWR3_(HC), consisting of somatically-mutated residues andextended by a four-residue insertion, reached like a thumb into thepocket formed by loops D, V5 and outer domain residues352_(gp120)-358_(gp120) (FIG. 16A,B and 17A, FIG. 4B). CDRH1 and CDRH3contacted the gp120 inner domain (FIG. 16B, FIG. 4B), contributing to a1287 Å² interface between the 8ANC195 HC and gp120 protein residues. TheCDRH1 and CDRH3 loop conformations, conserved in the free Fab (FIG. 16A,FIG. 2A,B), were necessary for binding gp120 since extending these loopswould result in clashes with gp120. The resulting antibody combiningsite was exquisitely suited to contacting portions of the inner domainof gp120 not targeted by other bNAbs (FIG. 16C).

The 8ANC195 Fab also made extensive interfaces with glycans attached toAsn234_(gp120) (buried surface area=1,653 Å²) and Asn276_(gp120) (buriedsurface area=731 Å²), rationalizing its dependence on these PNGSs forneutralization (West, Jr. et al., Proceedings of the National Academy ofSciences of the United States of America 110, 10598 (2013); Chuang etal., Journal of virology 87, 10047 (2013)). Together with CDRH2,somatically-mutated FWR residues in strands B, C″, D and E contributedto an extensive interface with the Asn234_(gp120)-associated N-glycan(usually high mannose in native HIV-1 Envs (Go et al., Journal ofvirology 85, 8270 (2011) that involved 10 sugar moieties, includingspecific interactions with terminal mannose residues (FIG. 17C,E, FIG.4C,D). A two-residue deletion at the CDRH2-FWR3_(HC) boundary comparedto the germline sequence permitted these interactions, since the longerloop would clash with inner domain residue Asn234_(gp120) and itsneighbors. The Asn276_(gp120) glycan (a complex-type N-glycan in nativeHIV-1 Envs (Go ct al., Journal of virology 85, 8270 (2011); Binley etal., Journal of virology 84, 5637 (2010)), but high mannose in thecrystallized gp120) was wedged between 8ANC195 and sCD4, where itcontacted FWR residues in strands A and B and the N-terminal portion ofCDRH1, forming an interface involving only the core pentasaccharidecommon to both high mannose and complex-type N-glycans (FIG. 17D, FIG.4E,F).

The 8ANC195 BC was bracketed by the Asn234_(gp120) and Asn276_(gp120)glycans in a manner analogous to interactions of HIV-1 antibodies thatpenetrate the Env glycart such as PG16 (interactions withAsn156_(gp120)/Asn173_(gp120) and Asn160_(gp120) glycans) (Pancera al.Nature structural & molecular biology 20, 804 (2013), PGT128 (withAsn301_(gp120) and Asn332_(gp120) glycans) (Pejchal et al., Science 334,1097 (2011)) and PGT121 (with Asn137_(gp120) and Asn332_(gp120) glycans)(Mouquet ct al., Proceedings of the National Academy of Sciences of theUnited States of America 109, E3268 (2012); Julien et al., Science 342,1477 (2013) Julien et al., PLoS pathogens 9, e1003342 (2013)) (FIG. 5).However, in contrast to these antibodies, 8ANC195 contacts with gp120were made exclusively by its HC; indeed, 33% of 8ANC195 V_(H) domainresidues not buried at the LC interface contacted gp120. In summary, the8ANC195-gp120 structure demonstrated that 8ANC195 recognizes a novelepitope involving the Asn234_(gp120) and Asn276_(gp120) glycans, thegp120 inner domain, loop D and loop V5, which would be adjacent to gp41in Env trimer (Julien et al., Science 342, 1477 (2013); Lyumkis et al.,Science 342, 1484 (2013)).

EXAMPLE 3 Negative Stain Single Particle Electron Microscopy (EM) toDetermine the Structure of 8ANC195 Fab Bound to a Soluble SOSIP Trimer

To investigate portions of the 8ANC195 epitope beyond the gp120 core,including potential contacts with gp41, negative stain single particleEM was used to determine the structure of 8ANC195 Fab bound to a solubleHIV-1 SOSIP trimer derived from strain BG505 (FIG. 6) (Julien et at.,Science 342, 1477 (2013): Lyumkis et al., Science 342, 1484 (2013);Sanders et al., PLoS pathogens 9, e1003618 (2013)). Independent dockingof the RG505 Env trimer structure (PUB 4NCO) (Julien et al., Science342, 1477 (2013)) and 8ANC195 Fab resulted in a model wherein the Fabcontacted both gp120 and gp41 within a single protomer (FIG. 18A, FIG.7). The EM model placed the CDRL1, CDRL2, and portions of FWR3_(LC) andCDRH3 in close proximity to the HR2 helix of gp41 (FIG. 18B). Althoughgp41 residues were not definitively identified in the trimer crystalstructure (Julien et al., Science 342, 1477 (2013)), based on theassignment of the HR2 C-terminus as Gly664_(gp41)(Lyumkis et. al.,Science 342, 1484 (2013), the kink in the HR2 helix was assigned asAsn637_(gp)41 (FIG. 18B, FIG. 8), the asparagine of a highly conservedPNGS. The EM model predicted that the Asn637_(gp41)-linked glycan andadjacent amino acid residues on HR2 interacted with 8ANC195 CDRH3, CDRL1and CDRL2.

Docking of the gp120-8ANC195 portion of the ternary crystal structureonto the SOSIP trimer structure resulted in a slightly different angularplacement of the Fab in the EM density than when the 8ANC195 Fab was fitindependently (FIG. 18A, FIG. 7). The Fab, especially the LC, was pushedfurther away from gp41 by comparison to the placement suggested by thecomplex crystal structure. The LC position in the EM model was morelikely to be accurate since it left space for bulky side chains atpositions 625_(gp41)-640_(gp41) that were modeled as alanines in thetrimer crystal structure (Julien et al., Science 342, 1477 (2013);Lyumkis et al., Science 342, 1484 (2013)). The slightly differentplacements could be due to crystal packing effects, spatial restraintsimposed by the gp41 glycans that were not present in the 8ANC195-gp120complex, removal of the PNGS at Asn88_(gp120) in the gp120 core, whichmay have allowed for a closer association of 8ANC195 and gp120 in thecrystal structure, and/or a small conformational change in the gp120region of the trimer to accommodate the Fab orientation trapped bycrystallization.

EXAMPLE 4 Neutralization and Binding Assays

The EM reconstruction highlighted a potential role for 8ANC195 LCcontacts to gp41. To assess LC contacts with trimeric Env, chimerasconsisting of the 8ANC195 HC paired with different. Leswere tested inneutralization and binding assays. The chimeras included a full germlineLC, a mature LC with individual CDR loops reverted to their germlinesequences or CDRL3 partially mutated to alanines, or the LC from the CD4binding site antibody 3BNC117 (FIG. 19A). As expected from the crystalstructure in which all gp120 contacts wore made by the 8ANC195 HC, thechimeras bound normally to gp120 core and to a full-length 93TH057 gp₁₂₀(FIG. 19B, table 3), thus changes in the LC did not disrupt the HCportion of the antibody combining site.

In contrast to gp120 binding, neutralization potencies assayed againstnative Env spike trimers were decreased by changes in the 8ANC195 LC.For example, reverting CDRL1 and CDRL2 sequences to germline precursorsequences (changing 3 of 7 and 3 of 3 residues, respectively) almostcompletely abrogated neutralization of YU2, 8ANC195-sensitive strain.Changes to CDRL3 led to a moderate reduction in neutralization potency,as did substituting the 3BNC117 LC for the cognate LC (FIG. 19B, table3). A chimeric IgG with one of the most conservatively-substituted LCs(Thr-Gly-Asn, mature CDRL1 containing a one-residue insertion, revertedto Ser-Ser, germline CDRL1) displayed unchanged binding to gp120, yetshowed reductions in neutralization potency of up to 250-fold.Similarly, conservative changes in CDRL2 (Arg-Gly-Ala, the mature CDRL2,reverted to the germline Lys-Ala-Ser sequence) caused large reductionsin neutralization potencies but had little effect on gp120 binding.Overall the data showed differential sensitivities of the binding andneutralization assays to changes in the 8ANC195 LC that were distantfrom the gp120 surface, which supported the EM results suggesting thatLC, and CDRL1 and CDRL2 in particular, contacted gp41

EXAMPLE 5 Isolation of Antibodies

To further investigate Env recognition by 8ANC195, additional members ofthis antibody clone were isolated from the original donor by single cellsorting using gp120 stabilized in the CD4-bound conformation (2CC core)as bait (FIG. 9), From 1536 single 2CC core-binding B cells, 10 (0.7%)were clonally related to 8ANC195, and of these, only four differedslightly from the two previously-described members (1 to 3 and 1 to 7residue differences in the HCs and LCs, respectively) (FIG. 10).Consistent with the limited sequence diversity, these antibodiesexhibited similar potencies to 8ANC195 in neutralization assays againsta panel of 15 Tier 2 viruses (FIG. 9C and Table 4).

TABLE 4 Virus 8ANC3040 8ANC3484 8ANC3630 8ANC3044 8ANC3430 8ANC195REJO4541.67 0.198 0.117 2.652 0.278 0.198 0.08 PVO.4 0.284 0.077 0.1020.260 0.206 0.52 YU2.DG 0.617 0.461 0.468 0.747 0.545 0.79 3415.v1.c13.059 0.589 27.977 7.557 >23 2.404 3365.v2.c20 >25 >30 >30 >30 >23 >30ZM53M.PB12 14.910 11.581 >30 15.164 >23 9.626 ZM109F.PB4NT >30 >30 >30 >23 >30 3016.v5.c45 0.427 0.131 0.136 0.242 0.271 0.195231965.c1 1.174 0.294 0.375 1.332 1.190 0.514 X1254_c3 2.909 2.192 2.3774.538 4.284 1.524 251-18 0.571 0.391 0.730 0.858 6.170 0.284 R1166.c12.370 1.027 1.453 2.381 3.642 0.986 H086.8 NT 0.394 0.300 3.830 >230.095 Du172.17 NT 4.011 >30 >30 >23 10.797 250-4 NT >30 >30 >30 >23 >50MuLV >30 >30 >30 >30 >23 >23Reasoning that the 2CC co e bait might fail to capture some 8ANC195family members, clone-specific primers were used to amplify 8ANC195variants from purified populations of CD19+ IgG+ memory B cells (FIG.11). 128 HC and 100 LC sequences were obtained that were clonallyrelated to 8ANC195 and displayed greater sequence diversity thanantibodies obtained using antigen-specific selection (FIGS. 10, 12). Ofthe 13 HC and 6 LC genes exhibiting greatest diversity, all combinationswere co-transfeeted in order to evaluate their neutralizing activityagainst a 15-member Tier 2 virus panel. 3 of 39 (7.7%) new antibodieswere at least as broad and potent as 8ANC195 (FIG. 19C and Table 5).

TABLE 5 γ3 γ4 γ8 γ15 γ20 γ22 γ23 γ44 γ46 γ52 γ59 Virus γ3κ3 γ4κ3 γ8κ3γ15κ3 γ20κ3 γ22κ3 γ52κ3 γ59κ3 REJO4541.07 0.260 >15 >15 >15 >15 >15 >158.543 PVO.4 0.170 >15 >15 5.316 >15 >15 1.918 >15 YU2.DG 0.420 >15 5.4101.068 >15 >15 5.751 8.970 34>15.v1.c1 >15 >15 >15 >15 >15 >15 >15 >153385.v2.c20 >15 >15 >15 >15 >15 >15 >15 >15ZM53M.PB12 >15 >15 >15 >15 >15 >15 >15 >15ZM109F.PB4 >15 >15 >15 >15 >15 >15 >15 >15 3016.v5.c45 0.347 >15 2.3831.265 >15 >15 3.050 >15 231965.c1 0.727 >15 3.477 1.611 >15 >156.594 >15 X1254_c3 1.822 18.047 4.009 3.487 14.702 >15 >15 3.392251-18 >15 >15 >15 >15 >15 >15 14.537 >15 R1160.c1 2.200 >15 7.5963.943 >15 >15 23.408 24.226 H088.8 3.307 >15 >15 >15 >15 >15 >15 >15Du172.17 >15 >15 >15 >15 >15 >15 >15 >15250-4 >15 >15 >15 >15 >15 >15 >15 >15 MuLV >30 NT >18 >30 >21 NT >30 >30Virus γ3κ5 γ22κ5 γ23κ5 γ46κ5 γ52κ5 γ59κ5 REJO4541.07 0.097 0.795 0.0910.196 0.035 4.669 PVO.4 0.081 0.352 0.043 0.129 0.019 >15 YU2.DG 0.2000.569 0.136 0.553 0.065 7.278 34>15.v1.c1 >15 >15 1.479 >15 0.120 >153365.v2.c20 >15 >15 >15 >15 >15 >15 ZM53M.PB12 >15 >15 5.875 12.4023.134 24.057 ZM109F.PB4 >15 >15 >15 >15 >15 >15 3016.v5.c45 0.314 0.1030.091 0.111 0.017 >15 231965.c1 0.697 0.690 0.291 0.525 0.094 >15X1254_c3 1.717 1.521 0.919 1.793 0.504 9.416 251-18 0.721 1.696 0.1760.609 0.048 6.959 R1106.c1 2.074 2.395 1.075 0.922 0.319 28.201 H080.80.434 >15 0.474 1.750 0.175 >15 Du172.17 3.728 >15 1.814 >15 NT >15250-4 >15 >15 >15 >15 >15 >15 MuLV >15 >30 >30 NT NT >30 Virus γ3κ11γ8κ11 γ15κ11 γ20κ11 γ22κ11 γ23κ11 γ44κ11 γ46κ11 γ52κ11 γ59κ11REJO4541.67 0.091 >15 >15 >15 >15 0.140 >15 >15 3.473 1.572 PVO.4 0.0748.163 6.704 >15 >15 0.103 >15 2.921 0.103 >15 YU2.DG 0.276 9.3494.122 >15 >15 0.340 >15 2.101 0.298 6.91134>15.v1.c1 >15 >15 >15 >15 >15 >15 >15 >15 >15 >153365.v2.c20 >15 >15 >15 >15 >15 >15 >15 >15 >15 >15ZM53M.PB12 >15 >15 >15 >15 >15 >15 >15 >15 >15 >15ZM109F.PB4 >15 >15 >15 >15 >15 >15 >15 >15 >15 >15 3016.v5.c45 0.1452.463 1.325 >15 >15 0.159 >15 1.932 0.161 >15 231965.c1 0.455 2.4905.103 >15 >15 0.543 >15 2.950 0.298 >15 X1254_c3 2.306 4.144 13.6877.503 >15 1.695 >15 5.682 2.419 4.669 251-18 >15 >15 >15 >15 >152.490 >15 >15 0.647 >15 R1166.c1 1.792 3.883 9.813 20.237 >15 1.611 >153.390 1.159 12.590 H088.6 0.778 >15 >15 >15 >15 >15 >15 >15 1.638 >15Du172.17 >15 >15 >15 >15 >15 11.298 >15 >15 >15 >15250-4 >15 >15 >15 >15 >15 >15 >15 >15 >15 >15 MuLV >30NT >30 >30 >30 >30 >19 >30 >30 >30 Virus γ3κ18 γ4κ18 γ15κ18 γ20κ18γ22κ18 γ23κ18 γ46κ18 γ52κ18 γ59κ18 REJO4541.67 0.049 >15 >15 >15 >150.061 >15 2.132 1.227 PVO.4 0.028 >15 1.588 >15 >15 0.047 2.1000.081 >15 YU2.DG 0.070 >15 1.169 >15 8.229 0.117 2.360 0.265 7.70734>15.v1.c1 >15 >15 >15 >15 >15 >15 >15 >15 >153365.v2.c20 >15 >15 >15 >15 >15 >15 >15 >15 >15ZM53M.PB12 >15 >15 >15 >15 >15 >15 >15 >15 >15ZM109F.PB4 >15 >15 >15 >15 >15 >15 >15 >15 >15 3016.v5.c45 0.057 >150.664 >15 13.350 0.047 2.993 0.129 >15 231965.c1 0.163 >15 1.491 >1525.988 0.128 4.157 0.284 >15 X1254_c3 0.616 16.224 2.849 5.221 1.5830.545 4.202 1.567 4.068 251-18 >15 >15 >15 >15 >15 1.578 >15 0.11625.039 R1168.c1 0.578 >15 1.986 20.036 7.311 0.708 4.096 1.351 11.701H088.8 0.209 >15 >15 >15 >15 >15 >15 0.464 >15 Du172.1711.518 >15 >15 >15 >15 2.953 >15 >15 >15250-4 >15 >15 >15 >15 >15 >15 >15 >15 >15 MuLV >30 >30NT >30 >30 >30 >15 >15 >15 Virus γ20κ19 REJO4541.67 >15 PVO.4 >15YU2.DG >15 34>15.v1.c1 >15 3365.v2.c20 >15 ZM53M.PB12 >15 ZM109F.PB4 >153016.v5.c45 >15 231965.c1 >15 X1254_c3 2.991 251-18 >15 R1166.c1 >15H088.8 >15 Du172.17 >15 250-4 >15 MuLV >15 Virus γ15κ61 γ44κ61 γ46κ61γ52κ61 γ59κ61 REJO4541.67 2.028 >15 0.378 0.115 1.333 PVO.4 1.230 >150.244 0.078 >15 YU2.DG 3.445 >15 0.918 0.408 4.32534>15.v1.c1 >15 >15 >15 0.681 >15 3365.v2.c20 >15 >15 >15 >15 >15ZM53M.PB12 >15 >15 27.493 8.435 13.301 ZM109F.PB4 >15 >15 >15 >15 >153016.v5.c45 1.214 >15 0.383 0.182 >15 231965.c1 5.231 >15 1.5760.516 >15 X1254_c3 10.884 >15 4.458 2.566 4.010 251-18 4.655 >15 1.8420.273 1.402 R1166.c1 6.548 >15 2.578 1.635 7.461 H088.8 >15 >15 0.5840.242 >15 Du172.17 >15 >15 26.083 7.275 >15 250-4 >15 >15 >15 >15 >15MuLV >15 >15 >15 >15 >15

Of these, δ52_(HC)κ5_(LC) was 5-fold more potent than 8ANC195(neutralized 12 of 15 viruses with a mean IC₅₀ of 0.45 μg/ml as comparedto 2,3 μg/ml for 8ANC195) (FIG. 13), a potency and breadth against thisvirus panel that was comparable to those of other bNAbs, such as VRCO(neutralized 12 of 15 viruses with a 0.56 μg/ml mean IC₅₀) and 10-1074(neutralized 6 of 15 viruses with a mean of 0.09 μg/ml), that targetnon-overlapping sites (Wu et al. Science 329, 856 (2010); Mouquet etal., Proceedings of the National Academy of Sciences of the United Stateof America 109, E3268 (2012)).

The LC was critical to the activity of more potent δ52_(HC)κ5_(LC)variant, as demonstrated by diminished neutralization potencies whenκ5_(LC) was swapped for either κ3_(LC) or κ11_(LC) (FIG. 19C). Theweaker neutralization could be explained by differences between κ5_(LC)and κ3_(LC) at solvent-exposed residues in CDRL2 (53_(LC) and 54_(LC))and FWRL3 (64LC), and a nearby buried residue (34_(LC) that may affectthe structural integrity of CDRL1, Modeling of YU2 gp41 residues intothe Env trimer structure (Julien et al., Science 342, 1477 (2013))suggested that 8ANC195 positions 53_(LC) and 54_(LC) were adjacent tothe Asn637_(gp41) PNGS (FIGS. 8, 14). The improved neutralizing activityof κ5_(LC) compared with the other newly-isolated LCs was associatedwith small side chains at positions 34_(LC) (Val), 53_(LC) (Ala) and54_(LC) (Ala), whereas κ3_(LC) or κ11_(LC), which were less broadlyneutralizing when paired with identical HCs, included bulkier and/orcharged side chains that would clash with the nearby gp41 glycan.κ5_(LC) was the only LC containing an S64R_(LC) substitution and thissingle change compared to the 8ANC195 LC may account for the 5-foldimproved potency of δ52_(HC)δ5_(LC). Residues in the immediate vicinityof Asn637_(gp41) might also modify neutralization; all six viral strainsthat were potently neutralized by the δ52_(HC)κ5_(LC) variant hadAsp636_(gp41) or Asn636_(gp41) whereas the remaining eight strains hadSer636_(gp41) (p<0,001 by G-test). The same association betweenAsp636_(gp41)/Asn636_(gp41) and neutralization potency was alsostatistically significant for 8ANC195 (p<0.01 by G-test), consistent aninteraction between the N-terminal portion of gp41 HR2 (residues ˜625 to640) and 8ANC195 LC (FIG. 8). Also consistent with changes in the gp41HR2 region affecting 8ANC195 neutralization, a computational analysis ofneutralization panel data using the Antibody Database program (West, Jr,et al., Proceedings of the National Academy of Sciences of the UnitedStates of America 110, 10598 (2013)) suggested that Glu632_(gp41) wasassociated with stronger neutralization.

In conclusion, 8ANC195 defines a novel site of HIV-1 Env vulnerabilityto neutralizing antibodies that spans gp120 and gp41 (FIG. 15). Ratherthan penetrating the glycan shield using only a single CDR loop, astrategy employed by antibodies such as PG9 and PGT128 (Pejchal et al.,Science 334, 1097 (2011); McLellan et al., Nature 480, 336 (2011))8ANC195 inserted its entire HC variable region into a gap in the shieldto form a large interface, of which >50% involved contacts to gp120glycans (FIG. 17).

Although it was not possible to obtain large numbers of 8ANC195 variantsby standard single cell cloning techniques (Scheid et al., J ImmunolMethods 343, 65 (2009)), randomly combining HCs and LCs obtained frommemory B cells without antigen-specific sorting demonstrated that thetarget of this antibody supported neutralization activity comparable tothat against the most vulnerable sites on Env, Potent variants of8ANC195 are particularly since the epitope does not overlap with thetargets of CD4 binding site, V2 loop, V3 loop or MPER antibodies.

The foregoing examples and description of the preferred embodimentsshould be taken as illustrating, rather than as limiting the presentinvention as defined by the claims. As will be readily appreciated,numerous variations and combinations of the features set forth above canbe utilized without departing from the present invention as set forth inthe claims. Such variations are not regarded as a departure from thescope of the invention, and all such variations are intended to beincluded within the scope of the following claims. All references citedherein are incorporated herein by reference in their entireties.

What is claimed is:
 1. An isolated anti-HIV-1 antibody comprising one orboth of a heavy chain comprising the sequence of any one of SEQ ID NOs:-1-18 and a light chain comprising the sequence of any one of SEQ IDNOs: 19-33.
 2. The isolated antibody of claim 1 wherein the heavy chaincomprises the sequence of SEQ ID NO: 1 and the light chain comprises thesequence of SEQ ID NO: 20,
 3. The isolated antibody of claim 1 whereinthe antibody is a human antibody.
 4. The isolated antibody of claim 1wherein the antibody is a chimeric antibody.
 5. An isolated polypeptidecomprising the sequence of any one of SEQ ID NOs: 1-33.
 6. An isolatednucleic acid encoding the antibody of claim 1 or the polypeptide ofClaim
 5. 7. A vector comprising the isolated nucleic acid of claim
 6. 8.A cultured cell comprising the vector of claim
 7. 9. A compositioncomprising the antibody of claim 1 or a fragment thereof that binds tothe 8ANC195 epitope of the HIV-1 envelope spike.
 10. A method ofpreventing or treating an HIV-1 infection in a subject in need thereofcomprising administering to said subject the composition of claim 9 inan amount effective to prevent or treat said HIV-1 infection.
 11. Anisolated antigen comprising an epitope-scaffold that mimics the HIV-1envelope spike epitope of broadly neutralizing antibody 8ANC195.
 12. Theantigen of claim 11 wherein the epitope-scaffold comprises adiscontinous epitope and a scaffold, wherein the epitope is derived fromHIV-1 gp120 and gp41, and wherein at least part of the scaffold is notderived from gp120 or gp41.
 13. The antigen of claim 12 wherein thediscontinuous epitope comprises amino acids corresponding to amino acidnumbers 44-47, 90-94, 97, 234, 236-238, 240, 274-278, 352-354, 357, 456,463. 466, 487, and 625-641 of gp140 from HIV strain 93TH057 numberedusing standard numbering for HIV strain HXBC2.
 14. The antigen of claim13 wherein the amino acids corresponding to amino acid numbers 234 and276 are glycosylated.
 15. An isolated nucleic acid encoding the antigenof claim
 11. 16. A vector comprising the isolated nucleic acid of claim15.
 17. A cultured cell comprising the vector of claim
 16. 18. Acomposition comprising the antigen of claim
 11. 19. A method forinducing an HIV antigen-specific immune response in a subject in needthereof, comprising administering to said subject the composition ofclaim 18 in an amount effective to generate an immune response.
 20. Amethod of preventing or treating an HIV-1 infection in a subject in needthereof comprising administering to said subject the composition ofclaim 19 in an amount effective to generate an immune response.
 21. Amethod for detecting or isolating an HIV-1 binding antibody in a subjectcomprising obtaining a biological sample from said subject, contactingsaid sample with the antigen of claim 11, and conducting an assay todetect or isolate an binding antibody.